Methods and compositions for the identification, assessment, and therapy of small cell lung cancer

ABSTRACT

The invention provides novel methods and compositions for modulating small cell lung cancer (SCLC) proliferation and metastasis through modulation of CXCR4 activity or expression. Also provided are methods for identifying compounds that modulate SCLC proliferation and metastasis through modulation of CXCR4 activity or expression. Further provided are methods for treating SCLC proliferation and metastasis, as well as methods for determining whether subjects are suitable candidates for treatment of SCLC via modulation of CXCR4 activity.

BACKGROUND OF THE INVENTION

Small cell lung cancer (SCLC) is an aggressive cancer withcharacteristic early metastasis. Even with the best available therapy,the overall survival for patients with SCLC is only 12% for earlydisease and 2% for metastatic disease. Metastatic disease is the usualpresentation for approximately two thirds of the patients with SCLC.Usual sites of metastasis include the liver, the adrenal glands, thebrain, the bone, and the bone marrow (Chute, J. P. et al. (1999) J.Clin. Oncol. 17:1794-801; Chute, J. P. et al. (1997) Mayo Clin. Proc.72:901-12; Salgia, R. (1996) Adv. Oncol. 12:8-15). Understanding themechanisms of metastasis in SCLC to the lymph nodes and various organsis crucial in designing future therapeutics.

SCLC is characterized by overexpression of several receptor tyrosinekinases (RTKs). Some of these RTKs are proto-oncogenes and keyregulators of cell growth, differentiation, survival, and motility.Recent work has begun to identify novel therapeutic agents for SCLC thattarget these RTKs (Gibbs, J. B. and Oliff, A. (1994) Cell 79:193-8;Levitzki, A. and Gazit, A. (1995) Science 267:1782-8). The c-Met andc-Kit RTKs have been identified as being important in SCLC. The c-Kitreceptor, a proto-oncogene product with a molecular weight of 145 kDa,is a class III RTK similar to c-Fms, Flt3, and platelet derived growthfactor receptor (PDGFR). The c-Kit protein contains fiveimmunoglobulin-like domains in the extracellular regions, atransmembrane domain, and a cytoplasmic domain with two kinase domainsseparated by a kinase insert (Ashman, L. K. (1999) Int. J. Biochem. CellBiol. 31:1037-51; Linnekin, D. (1999) Int. J. Biochem. Cell Biol.31:1053-74; Matthews, W. et al., (1991) Cell 65:1143-52). Approximately70% of SCLC tumor specimens and cell lines coexpress c-Kit and itsnatural ligand, stem cell factor (SCF). The SCF/c-Kit pathway isfunctional in an autocrine or a paracrine fashion in SCLC (Hibi, K. etal. (1991) Oncogene 6:2291-6; Krystal, G. W. et al. (1996) Cancer Res.56:370-6; Plummer, H., 3^(rd) et al. (1993) Cancer Res. 53:4337-42;Rygaard, K. et al. 91993) Br. J. Cancer 67:37-46; Sekido, Y. et al.(1991) Cancer Res. 51:2416-9; Sekido, Y. et al. (1993) Cancer Res.53:1709-14; Yamanishi, Y. et al. (1996) Jpn. J. Cancer Res. 87:53442.The c-Kit receptor can be inhibited by a variety of inhibitors,including the novel tyrosine kinase inhibitor imatinib mesylate (alsoreferred to as STI571 or Gleevec™ (Krystal, G. W. et al. (1997) CancerRes. 57:2203-8; Krystal, G. W. et al. (2001) Cancer Res. 61:3660-8;Krystal, G. W. et al. (2000) Clin. Cancer Res. 6:3319-26; Tuveson, D. Aet al. (2001) Oncogene 20:5054-8; Wang, W. L. et al. (2000) Oncogene3521-8)). STI571, having the chemical name4-(4-methylpiperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl)-pyrimidin-2-ylamino)-phenyl]-benzamide,is further described in EP 0 564 409 and, in the form of the methanesulfonate salt, especially in the preferred β-crystal form, in WO99/03854.

In hematopoietic cells, the c-Kit receptor has been shown tofunctionally interact with a variety of molecules, including chemokinereceptors (Dutt, P. et al. (1998) J. Immunol. 161:3652-8; Kim, C. H. andBroxmeyer, H. E. (1998) Blood 91:100-10; Lataillade, J. J. et al. (2000)Blood 95:756-68; Peled, A. et al. (1999) Science 283:845-8) (13, 21, 26,33). Given the lack of existing therapies to treat SCLC, particularlymetastatic SCLC, there exists a need to identify additional moleculartargets that may be used in SCLC for the identification of noveltherapies.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for theidentification, assessment, and therapy of small cell lung cancer(SCLC). The present invention is based, at least in part, on thediscovery that CXCR4 is ubiquitously expressed, and c-Kit is variablyexpressed, in SCLC cells. The present invention is further based on thediscovery that treatment of SCLC cells with stromal cell derivedfactor-1α (SDF-1α) and/or stem cell factor (SCF) (the ligands for CXCR4and c-Kit, respectively) induces proliferation and regulates adhesion,motility, and cell shape of SCLC cells. The present invention is stillfurther based on the discovery that PI3-Kinase (PI3-K) regulates SDF-1αinduced cell motility of SCLC cells, that CXCR4- and c-Kit cooperativelyinduce morphological changes in SCLC cells, that SDF-1α and SCFindependently regulate phosphorylation of Akt and p70 S6 kinase, andthat STI571 (a c-Kit inhibitor also referred to as imatinib mesylate orGleevec™) and LY294002 (a PI3-K inhibitor) inhibit signal transductionof the CXCR4 and c-Kit pathways.

Accordingly, in one embodiment, the invention provides methods ofinhibiting cellular proliferation, cellular movement or motility, andmorphological change in a small cell lung cancer cell population,comprising contacting the population with a CXCR4 inhibitor. In anotherembodiment the invention provides methods of modulating cellularadhesion in a small cell lung cancer cell population comprisingcontacting the population with a CXCR4 inhibitor. In a preferredembodiment, PI3-K activity in the small cell lung cancer cell populationis down-regulated.

In one embodiment, the CXCR4 inhibitor binds to CXCR4 or to SDF-1α. Inanother embodiment, the CXCR4 inhibitor is an antibody or antibodyfragment. In still another embodiment, the CXCR4 inhibitor is a smallmolecule, for example, AMD-3100, ALX40-4C, T22, T140, Met-SDF-1beta,T134, or AMD-3465. In another embodiment, the methods of the inventionfurther comprise contacting the small cell lung cancer cell populationwith a receptor tyrosine kinase inhibitor, for example, a c-Kitinhibitor such as imatinib mesylate (Gleevec™).

In another embodiment, the invention provides methods of treating asubject with small cell lung cancer comprising administering a CXCR4inhibitor to the subject. In one embodiment, the CXCR4 inhibitor bindsto CXCR4 or to SDF-1α. In another embodiment, the CXCR4 inhibitor is anantibody or antibody fragment. In still another embodiment, the CXCR4inhibitor is a small molecule, for example, AMD-3100, ALX40-4C, T22,T140, Met-SDF-1beta, T134, or AMD-3465. In another embodiment, themethods further comprise administering a receptor tyrosine kinaseinhibitor, for example, a c-Kit inhibitor such as imatinib mesylate(Gleevec™), to the subject. In a preferred embodiment, PI3-K activity inthe small cell lung cancer cells of the subject is down-regulated.

In another embodiment, the invention provides methods of inhibitingmetastasis of small cell lung cancer in a subject comprisingadministering a CXCR4 inhibitor to the subject. In one embodiment, theCXCR4 inhibitor binds to CXCR4 or to SDF-1α. In another embodiment, theCXCR4 inhibitor is an antibody or antibody fragment. In still anotherembodiment, the CXCR4 inhibitor is a small molecule, for example,AMD-3100, ALX40-4C, T22, T140, Met-SDF-1beta, T134, or AMD-3465. Inanother embodiment, the methods further comprise administering areceptor tyrosine kinase inhibitor, for example, a c-Kit inhibitor suchas imatinib mesylate (Gleevec™), to the subject. In a preferredembodiment, PI3-K activity in the small cell lung cancer cells of thesubject is down-regulated.

In another embodiment, the invention provides methods for identifyingagents which can be used to treat small cell lung cancer comprisingdetermining whether the agent inhibits CXCR4, for example, bydetermining whether the agent can compete with SDF-1α for binding toCXCR4, whether the agent can inhibit SDF-1α mediated cellularproliferation, adhesion, motility, or cell shape, and/or whether theagent inhibits PI3-K activity (e.g., phosphorylation of Akt and p70 S6kinase).

In another embodiment, the invention provides a method for determiningwhether a CXCR4 inhibitor can or cannot be used to treat small cell lungcancer comprising obtaining a sample of lung cancer cells (e.g., from acell line or a subject), and determining whether the cells express CXCR4(e.g., by measuring CXCR4 mRNA and/or protein levels), therebydetermining that the CXCR4 inhibitor can be used to treat small celllung cancer when CXCR4 is expressed. In one embodiment, the CXCR4inhibitor may bind to CXCR4 or SDF-1α (e.g., an antibody, an antibodyfragment, or a small molecule such as AMD-3100, ALX40-4C, T22, T140,Met-SDF-1beta, T134, or AMD-3465).

In another embodiment, the invention provides a method for determiningwhether a combination of a CXCR4 inhibitor and a receptor tyrosinekinase inhibitor can or cannot be used to treat small cell lung cancercomprising obtaining a sample of lung cancer cells (e.g., from a cellline or a subject), and determining whether the cells express CXCR4(e.g., by measuring CXCR4 mRNA and/or protein levels) and furtherdetermining whether the cells also express the receptor tyrosine kinasethat is inhibited by said receptor tyrosine kinase inhibitor (e.g., bymeasuring mRNA and/or protein levels of the receptor tyrosine kinase),thereby determining that the combination of a CXCR4 inhibitor and areceptor tyrosine kinase inhibitor can be used to treat small cell lungcancer when CXCR4 and the receptor tyrosine kinase are expressed. In apreferred embodiment of this method, the receptor tyrosine kinaseinhibitor is a c-Kit inhibitor such as for example imatinib mesylate(Gleevec™). The CXCR4 inhibitor may bind to CXCR4 or SDF-1α (e.g., anantibody, an antibody fragment, or a small molecule such as AMD-3100,ALX40-AC, T22, T140, Met-SDF-1beta, T134, or AMD-3465).

In another embodiment, the invention provides a method for determiningwhether a CXCR4 inhibitor can or cannot be used to inhibit proliferationor metastasis of small cell lung cancer comprising obtaining a sample oflung cells (e.g., from a cell line or a subject), and determiningwhether the cells express CXCR4 (e.g., by measuring CXCR4 mRNA and/orprotein levels), thereby determining that the CXCR4 inhibitor can beused to inhibit proliferation or metastasis of small cell lung cancerwhen CXCR4 is expressed. In one embodiment, the CXCR4 inhibitor may bindto CXCR4 or SDF-1α (e.g., an antibody, an antibody fragment, or a smallmolecule such as AMD-3100, ALX40-4C, T22, T140, Met-SDF-1beta, T134, orAMD-3465).

In another embodiment, the invention provides a method for determiningwhether a combination of a CXCR4 inhibitor and a receptor tyrosinekinase inhibitor can or cannot be used to inhibit proliferation ormetastasis of small cell lung cancer comprising obtaining a sample oflung cells (e.g., from a cell line or a subject), and determiningwhether the cells express CXCR4 (e.g., by measuring CXCR4 mRNA and/orprotein levels) and further determining whether the cells also expressthe receptor tyrosine kinase that is inhibited by said receptor tyrosinekinase inhibitor (e.g., by measuring mRNA and/or protein levels of thereceptor tyrosine kinase), thereby determining that the combination of aCXCR4 inhibitor and a receptor tyrosine kinase inhibitor can be used toinhibit proliferation or metastasis of small cell lung cancer when CXCR4and the receptor tyrosine kinase are expressed. In a preferredembodiment of this method, the receptor tyrosine kinase inhibitor is ac-Kit inhibitor such as for example imatinib mesylate (Gleevec™). TheCXCR4 inhibitor may bind to CXCR4 or SDF-1α (e.g., an antibody, anantibody fragment, or a small molecule such as AMD-3100, ALX40-4C, T22,T140, Met-SDF-1beta, T134, or AMD-3465).

In another embodiment, the invention provides a method for determiningwhether a patient would benefit from treatment with an agent thatinhibits CXCR4 comprising obtaining a lung sample from the patient anddetermining whether CXCR4 is expressed in the sample (e.g., by measuringCXCR4 mRNA and/or protein levels).

In another embodiment, the invention provides a method for determiningwhether a patient would benefit from treatment with an agent thatinhibits CXCR4 and an agent that inhibits c-Kit comprising obtaining alung sample from the patient and determining whether CXCR4 and c-Kit areexpressed in the sample (e.g., by measuring CXCR4 and c-Kit mRNA and/orprotein levels).

In another embodiment, the invention provides a method for determiningwhether treatment with a CXCR4 inhibitor should be continued ordiscontinued in a small cell lung cancer patient, comprising obtainingtwo or more samples comprising tumor cells from a patient during thecourse of treatment, determining the level of activity in the tumorcells of CXCR4, and continuing treatment when the activity of CXCR4 doesnot increase during treatment. In a preferred embodiment, the level ofactivity in the tumor cells of CXCR4 is determined by determining theability of SDF-1α to modulate proliferation, adhesion, motility, cellshape, or PI3-K activity in the cells.

In another embodiment, the invention provides a method for determiningwhether treatment with a combination of a CXCR4 inhibitor and a receptortyrosine kinase inhibitor should be continued or discontinued in a smallcell lung cancer patient, comprising obtaining two or more samplescomprising tumor cells from a patient during the course of treatment,determining the level of activity in the tumor cells of CXCR4,determining the level of activity in the tumor cells of c-Kit, andcontinuing treatment when the activity levels of CXCR4 and/or c-Kit donot increase during treatment. In a preferred embodiment, the level ofactivity in the tumor cells of CXCR4 is determined by determining theability of SDF-1α to modulate proliferation, adhesion, motility, cellshape, or PI3-K activity in the cells. In a further preferredembodiment, the level of activity in the tumor cells of c-Kit isdetermined by determining the ability of SCF to modulate proliferation,adhesion, motility, cell shape, or PI3-K activity in the cells.

In another embodiment, the invention provides a CXCR4 inhibitor for usein a method for the treatment of a subject. In one embodiment, the CXCR4inhibitor may bind to CXCR4 or SDF-1α (e.g., an antibody, an antibodyfragment, or a small molecule such as AMD-3100, ALX40-4C, T22, T140,Met-SDF-1beta, T134, or AMD-3465).

In another embodiment, the invention provides the use of a CXCR4inhibitor for the preparation of a pharmaceutical composition for use ina method such as inhibiting cellular proliferation in a small cell lungcancer cell population, inhibiting cellular movement or motility in asmall cell lung cancer cell population, modulating cellular adhesion ina small cell lung cancer cell population, inhibiting morphologicalchange in a small cell lung cancer cell population, treating a subjecthaving small cell lung cancer, or inhibiting metastasis of small celllung cancer in a subject. In one embodiment, the CXCR4 inhibitor maybind to CXCR4 or SDF-1α (e.g., an antibody, an antibody fragment, or asmall molecule such as AMD-3100, ALX40-4C, T22, T140, Met-SDF-1beta,T134, or AMD-3465).

In another embodiment, the invention provides a combination whichcomprises (a) a CXCR4 inhibitor and (b) a receptor tyrosine kinaseinhibitor, such as especially a c-Kit inhibitor, wherein the activeingredients (a) and (b) are present in each case in free form or in theform of a pharmaceutically acceptable salt, for simultaneous,concurrent, separate or sequential use, especially in a method for thetreatment of a subject, preferably in a method such as inhibitingcellular proliferation in a small cell lung cancer cell population,inhibiting cellular movement or motility in a small cell lung cancercell population, modulating cellular adhesion in a small cell lungcancer cell population, inhibiting morphological change in a small celllung cancer cell population, treating small cell lung cancer, orinhibiting metastasis of small cell lung cancer. In a preferredembodiment of this method, the receptor tyrosine kinase inhibitor is ac-Kit inhibitor such as for example imatinib mesylate (Gleevec™). TheCXCR4 inhibitor may bind to CXCR4 or SDF-1α (e.g., an antibody, anantibody fragment, or a small molecule such as AMD-3100, ALX40-4C, T22,T140, Met-SDF-1beta, T134, or AMD-3465).

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery thatCXCR4 is ubiquitously expressed, and c-Kit is variably expressed, insmall cell lung cancer (SCLC) cells. The present invention is furtherbased on the discovery that treatment of SCLC cells with SDF-1α and/orSCF (the ligands for CXCR4 and c-Kit, respectively) inducesproliferation and regulates adhesion, motility, and cell shape of SCLCcells. The present invention is still further based on the discoverythat PI3-Kinase (PI3-K) regulates SDF-1α induced cell motility of SCLCcells, that CXCR4— and c-Kit cooperatively induce morphological changesin SCLC cells, that SDF-1α and SCF independently regulatephosphorylation of Akt and p70 S6 kinase, and that STI571 (a c-Kitinhibitor also referred to as imatinib mesylate or Gleevec™) andLY294002 (a PI3-K inhibitor) inhibit signal transduction of the CXCR4and c-Kit pathways.

Chemokines are small cytokine-like peptides that direct various subsetsof hematopoietic cells to home specific anatomical sites throughinteraction with their G protein-coupled receptors (Rossi, D. andZlotnick, A. (2000) Annu. Rev. Immunol. 18:217-42; Zlotnick, A. andYoshie, O. (2000) Immunity 12:121-7). CXCR4 is a seven-transmembrane Gprotein-coupled receptor and is also known as a coreceptor for humanimmunodeficiency virus (HIV) (Bleul, C. C. et al. (1996) Nature382:829-3; Feng, Y. et al. (1996) Science 272:872-7; Nagasawa, T. et al.(1996) Proc. Natl. Acad. Sci. USA 93:14726-9). SDF-1α, the naturalligand for CXCR4, is a member of the CXC chemokine family which haschemotactic activity for hematopoietic progenitor cells (Aiuti, A. etal. (1997) J. Exp. Med. 185:111-20; Jo, D. Y. et al. (2000) J. Clin.Invest. 105:101-11; Nagasawa, T. et al. (1994) Proc. Natl. Acad. Sci.USA 91:2305-9; Nagasawa, T. (1996) Proc. Natl. Acad. Sci. USA93:14726-9). Thus far, the role of interaction between chemokinereceptors and cytokine receptors has not been defined for solid tumorssuch as SCLC.

The instant invention therefore provides methods and compositions forthe identification, assessment, and therapy of SCLC. In particular, thepresent invention provides methods and compositions for modulating SCLCgrowth, proliferation, movement, motility, adhesion, morphologicalchange, and/or metastasis by modulating CXCR4 activity in SCLC cells. Insome embodiments, the activity of c-Kit may also be modulated.Accordingly, one aspect of the invention pertains to the use of CXCR4and/or c-Kit molecules, referred to herein as CXCR4 and/or c-Kit nucleicacid and protein molecules, which comprise a family of molecules havingcertain conserved structural and functional features, and which play arole in or function in type I muscle formation associated activities.The term “family” when referring to the protein and nucleic acidmolecules of the invention is intended to mean two or more proteins ornucleic acid molecules having a common structural domain and havingsufficient amino acid or nucleotide sequence homology as defined herein.Such family members can be naturally occurring and can be from eitherthe same or different species. For example, a family can contain a firstprotein of human origin, as well as other, distinct proteins of humanorigin or alternatively, can contain homologues of non-human origin.Members of a family may also have common functional characteristics.

Another aspect of the invention pertains to methods for treating asubject having SCLC. These methods include the step of administering aCXCR4 modulator and/or a c-Kit modulator to the subject such thattreatment occurs. In a preferred embodiment, the subject being treatedwith a CXCR4 modulator has SCLC cells that express CXCR4. In a furtherpreferred embodiment, a subject being treated with a CXCR4 modulator anda c-Kit modulator has SCLC cells that express both CXCR4 and c-Kit. Theterms “treating” or “treatment, as used herein, refer to reduction oralleviation of at least one adverse effect or symptom of SCLC, e.g.,tumor burden, tumor size, tumor cell proliferation, migration, motility,adhesion, and/or morphological change.

As used herein, a CXCR4 modulator is a molecule which can modulate CXCR4nucleic acid expression and/or CXCR4 protein activity. For example, aCXCR4 modulator can modulate, i.e., upregulate (activate) ordownregulate (suppress), CXCR4 nucleic acid expression. In anotherexample, a CXCR4 modulator can modulate (i.e., stimulate or inhibit)CXCR4 protein activity. In a preferred embodiment, the methods of theinvention use CXCR4 inhibitors, i.e., agents which inhibit CXCR4activity. Non-limiting examples of CXCR4 inhibitors include smallmolecules such as AMD-3100, ALX40-4C, T22, T140, Met-SDF-1beta, T134,and AMD-3465, or small molecules or drugs identified by the screeningmethods described herein. CXCR4 inhibitors may also inhibit CXCR4nucleic acid expression, for example, antisense molecules, i.e., aribozyme, as described herein. Examples of antisense molecules which canbe used to inhibit CXCR4 nucleic acid expression include antisensemolecules which are complementary to a portion of the 5′ untranslatedregion of SEQ ID NO:1 which also includes the start codon and antisensemolecules which are complementary to a portion of the 3′ untranslatedregion of SEQ ID NO:1. A CXCR4 modulator which inhibits CXCR4 nucleicacid expression can also be a small molecule or other drug, i.e., asmall molecule or drug identified using the screening assays describedherein, which inhibits CXCR4 nucleic acid expression. A CXCR4 moleculeof the invention can thus also be used as a target to screen molecules,i.e., which can modulate CXCR4 activity. CXCR4 modulators may also beantibodies or antibody fragments. CXCR4 modulators may also be forms ofSDF-1α which block or inhibit CXCR4 activity, e.g., dominant negativeforms of SDF-1α.

As used herein, a c-Kit modulator is a molecule which can modulate c-Kitnucleic acid expression and/or c-Kit protein activity. For example, ac-Kit modulator can modulate, i.e., upregulate (activate) ordownregulate (suppress), c-Kit nucleic acid expression. In anotherexample, a c-Kit modulator can modulate (i.e., stimulate or inhibit)c-Kit protein activity. In a preferred embodiment, the methods of theinvention use c-Kit inhibitors, i.e., agents which inhibit c-Kitactivity. Non-limiting examples of CXCR4 inhibitors include smallmolecules or drugs identified by the screening methods described herein.In a preferred embodiment, a c-Kit inhibitor is a receptor tyrosinekinase inhibitor such as imatinib mesylate, also referred tointerchangeably herein as STI571 or Gleevec™ (available from Novartis(Basel, Switzerland)) (Savage, D. G. and Antman, K. H. (2002) N. Engl.J. Med. 346(9):683-93; Mauro, M. J. et al. (2002) J. Clin. Oncol.20(1):325-34; Schiffer, C. A. (2001) Semin. Oncol. 28(5 Suppl 17):34-9;Demetri, G. D. (2001) Semin. Oncol. 28(5 Suppl 17):19-26; Griffin, J.(2001) Semin. Oncol. 28(5 Suppl 17):3-8; Verweij, J. et al. (2001) Eur.J. Cancer. 37(15):1816-9; Shah, N. P. and Sawyers, C. L. (2001) Curr.Opin. Investig. Drugs. 2(3):422-3).

c-Kit inhibitors may also inhibit c-Kit nucleic acid expression, forexample, antisense molecules, i.e., a ribozyme, as described herein.Examples of antisense molecules which can be used to inhibit c-Kitnucleic acid expression include antisense molecules which arecomplementary to a portion of the 5′ untranslated region of SEQ ID NO:3which also includes the start codon and antsense molecules which arecomplementary to a portion of the 3′ untranslated region of SEQ ID NO:3.A c-Kit modulator which inhibits c-Kit nucleic acid expression can alsobe a small molecule or other drug, i.e., a small molecule or drugidentified using the screening assays described herein, which inhibitsc-Kit nucleic acid expression. A c-Kit molecule of the invention canthus also be used as a target to screen molecules, i.e., which canmodulate c-Kit activity. c-Kit modulators may also be antibodies orantibody fragments. c-Kit modulators may also be forms of SCF whichblock or inhibit c-Kit activity, e.g., dominant negative forms of SCF.

Other aspects of the invention pertain to methods for modulating a cellassociated activity. These methods include contacting the cell with anagent (or a composition which includes an effective amount of an agent)which modulates CXCR4 and/or c-Kit protein activity or CXCR4 and/orc-Kit nucleic acid expression such that a cell associated activity isaltered relative to a cell associated activity of the cell in theabsence of the agent. As used herein, “a cell associated activity”refers to a normal or abnormal activity or function of a cell, e.g., anSCLC cell. Examples of cell associated activities include proliferation,growth, movement, motility, adhesion, and/or morphological change. In apreferred embodiment, the cell associated activity is metastasis. Theterm “altered” as used herein refers to a change, i.e., an increase ordecrease, of a cell associated activity. In a preferred embodiment, theagent inhibits CXCR4 and/or c-Kit protein activity or CXCR4 and/or c-Kitnucleic acid expression. Examples of such inhibitory agents include anucleic acid molecule encoding a dominant negative CXCR4 and/or c-Kitprotein, a dominant negative SDF-1α and/or SCF protein, an antisenseCXCR4 and/or c-Kit nucleic acid molecule, an anti-CXCR4 and/oranti-c-Kit antibody or antibody fragment, and a modulatory agent whichinhibits CXCR4 and/or c-Kit protein activity or and/or c-Kit CXCR4nucleic acid expression and which may be identified using the drugscreening assays described herein. These modulatory methods can beperformed in vitro (i.e., by culturing the cell with the agent) or,alternatively, in vivo (i.e., by administering the agent to a subject).In a preferred embodiment, the modulatory methods are performed in vivo,i.e., the cell is present within a subject, i.e., a mammal, i.e., ahuman, and the subject has SCLC.

The methods of the present invention may therefore: 1) modulate growthof SCLC cells; 2) modulate proliferation of SCLC cells; 3) modulatemovement of SCLC cells; 4) modulate motility of SCLC cells; 5) modulateadhesion of SCLC cells; 6) modulate cellular shape and/or morphologicalchange of SCLC cells; 7) modulate metastasis of SCLC cells; 8) modulateCXCR4 activity; 9) modulate CXCR4 and c-Kit activity; 10) modulate CXCR4binding to SDF-1α; 11) modulate c-Kit binding to SCF; and/or 12)modulate PI3-K activity.

A nucleic acid molecule, a protein, a CXCR4 and/or c-Kit modulator, acompound etc. used in the methods of treatment can be incorporated intoan appropriate pharmaceutical composition described herein andadministered to the subject through a route which allows the molecule,protein, modulator, or compound etc. to perform its intended function.Examples of routes of administration are also described herein.

The nucleotide sequence of the human CXCR4 cDNA and the predicted aminoacid sequence of the human CXCR4 protein are shown in SEQ ID NOs:1 and2, respectively. The human CXCR4 gene, which is approximately 1679nucleotides in length, encodes a full length protein having a molecularweight of approximately 38.7 kD and which is approximately 352 aminoacid residues in length. Further description of the human CXCR4 nucleicacid and polypeptide sequences can be found in GenBank Accession Nos.NM_(—)003467 and NP_(—)003458, respectively; as well as in Federsppiel,B. et al. (1993) Genomics 16(3):707-712; Herzog, H. et al. (1993) DNACell Biol. 12(6):465-471; Jazin, E. E. et al. (1993) Regul. Pept47(3):247-258; Nomura, H. et al. (1993) Int. Immunol. 5(10):1239-1249;Loetscher, M. et al. (1994) J. Biol. Chem. 269(1):232-237; Moriuchi, M.et al. (1997) J. Immunol. 159(9):4322-4329; Caruz, A. et al. (1998) FEBSLett. 426(2):271-278; and Wegner, S. A. et al. (1998) J. Biol. Chem.273(8):4754-4760.

The nucleotide sequence of the human c-Kit cDNA and the predicted aminoacid sequence of the human c-Kit protein are shown in SEQ ID NOs:3 and4, respectively. The human c-Kit gene, which is approximately 5084nucleotides in length, encodes a full length protein having a molecularweight of approximately 107.4 kD and which is approximately 976 aminoacid residues in length. Further description of the human c-Kit nucleicacid and polypeptide sequences can be found in GenBank Accession Nos.NM_(—)000222 and NP_(—)000213, respectively; as well as in Yarden, Y. etal. (1987) EMBO J. 6(11):3341-3351; Andre, C. et al. (1992) Oncogene7(4):685-691; Giebel, L. B. et al. (1992) Oncogene 7(11):2207-2217;Yamamoto, K. et al. (1993) Jpn. J. Cancer Res. 84(11):1136-1144; Toyota,M. et al. (1994) Cancer Res. 54(1):272-275; Zhu W. M. et al. (1994)Leuk. Lymphoma 12(5-6):441-447; and Andre, C. (1997) Genomics39(2):216-226.

Various aspects of the invention are described in further detail in thefollowing subsections:

I. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to methods utilizing isolatednucleic acid molecules that encode CXCR4 and/or c-Kit, or biologicallyactive portions thereof, as well as nucleic acid fragments sufficientfor use as hybridization probes to identify CXCR4 and/or c-Kit encodingnucleic acid (i.e., CXCR4 and/or c-Kit mRNA). As used herein, the term“nucleic acid molecule” is intended to include DNA molecules (i.e., cDNAor genomic DNA) and RNA molecules (i.e., mRNA) and analogs of the DNA orRNA generated using nucleotide analogs. The nucleic acid molecule can besingle-stranded or double-stranded, but preferably is double-strandedDNA. An “isolated” nucleic acid molecule is one which is separated fromother nucleic acid molecules which are present in the natural source ofthe nucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. For example, invarious embodiments, the isolated CXCR4 and/or c-Kit nucleic acidmolecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5kb or 0.1 kb of nucleotide sequences which naturally flank the nucleicacid molecule in genomic DNA of the cell from which the nucleic acid isderived (i.e., a lung cell). Moreover, an “isolated” nucleic acidmolecule, such as a cDNA molecule, can be substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or chemical precursors or other chemicals when chemicallysynthesized.

A nucleic acid molecule of the present invention, i.e., a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO:1 or 3 or anucleotide sequence which is at least about 50%, preferably at leastabout 60%, more preferably at least about 70%, yet more preferably atleast about 80%, still more preferably at least about 90%, and mostpreferably at least about 95% or more homologous to the nucleotidesequence shown in SEQ ID NO:1 or 3 or a portion thereof (e.g., 50, 100,200, 300, 400, 450, 500, or more nucleotides), can be isolated usingstandard molecular biology techniques and the sequence informationprovided herein. For example, a human CXCR4 and/or c-Kit cDNA can beisolated from a human SCLC cell line (from Stratagene, LaJolla, Calif.,or Clontech, Palo Alto, Calif.) using all or portion of SEQ ID NO:1 or 3as a hybridization probe and standard hybridization techniques (i.e., asdescribed in Sambrook, J., Fritsh, E. F., and Maniatis, T. MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).Moreover, a nucleic acid molecule encompassing all or a portion of SEQID NO:1 or a nucleotide sequence which is at least about 50%, preferablyat least about 60%, more preferably at least about 70%, yet morepreferably at least about 80%, still more preferably at least about 90%,and most preferably at least about 95% or more homologous to thenucleotide sequence shown in SEQ ID NO:1 or 3 can be isolated by thepolymerase chain reaction using oligonucleotide primers designed basedupon the sequence of SEQ ID NO:1 or 3 or the homologous nucleotidesequence. For example, mRNA can be isolated from lung cells or lungtumor cells (i.e., by the guanidinium-thiocyanate extraction procedureof Chirgwin et al. (1979) Biochemistry 18:5294-5299) and cDNA can beprepared using reverse transcriptase (i.e., Moloney MLV reversetranscriptase, available from Gibco/BRL, Bethesda, Md.; or AMV reversetranscriptase, available from Seikagaku America, Inc., St. Petersburg,Fla.). Synthetic oligonucleotide primers for PCR amplification can bedesigned based upon the nucleotide sequence shown in SEQ ID NO:1 or 3 orto the homologous nucleotide sequence. A nucleic acid of the inventioncan be amplified using cDNA or, alternatively, genomic DNA, as atemplate and appropriate oligonucleotide primers according to standardPCR amplification techniques. The nucleic acid so amplified can becloned into an appropriate vector and characterized by DNA sequenceanalysis. Furthermore, oligonucleotides corresponding to a CXCR4 and/orc-Kit nucleotide sequence can be prepared by standard synthetictechniques, i.e., using an automated DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises the nucleotide sequence shown in SEQ ID NO:1 or 3 ora nucleotide sequence which is at least about 50%, preferably at leastabout 60%, more preferably at least about 70%, yet more preferably atleast about 80%, still more preferably at least about 90%, and mostpreferably at least about 95% or more homologous to the nucleotidesequence shown in SEQ ID NO:1 or 3. The sequence of SEQ ID NO:1corresponds to the human CXCR4 cDNA. This cDNA comprises sequencesencoding the CXCR4 protein (i.e., “the coding region”, from nucleotides89 to 1144), as well as 5′ untranslated sequences (nucleotides 1 to 88)and 3′ untranslated sequences (nucleotides 1145 to 1679). Alternatively,the nucleic acid molecule can comprise only the coding region of SEQ IDNO:1 (i.e., nucleotides 89 to 1144). The sequence of SEQ ID NO:3corresponds to the human c-Kit cDNA. This cDNA comprises sequencesencoding the c-Kit protein (i.e., “the coding region”, from nucleotides22 to 2949), as well as 5′ untranslated sequences (nucleotides 1 to 21)and 3′ untranslated sequences (nucleotides 2950 to 5084). Alternatively,the nucleic acid molecule can comprise only the coding region of SEQ IDNO:3 (i.e., nucleotides 22 to 2949).

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which is a complement ofthe nucleotide sequence shown in SEQ ID NO:1 or 3 or a nucleotidesequence which is at least about 50%, preferably at least about 60%,more preferably at least about 70%, yet more preferably at least about80%, still more preferably at least about 90%, and most preferably atleast about 95% or more homologous to the nucleotide sequence shown inSEQ ID NO:1 or 3. A nucleic acid molecule which is complementary to thenucleotide sequence shown in SEQ ID NO:1 or 3 or to a nucleotidesequence which is at least about 50%, preferably at least about 60%,more preferably at least about 70%, yet more preferably at least about80%, still more preferably at least about 90%, and most preferably atleast about 95% or more homologous to the nucleotide sequence shown inSEQ ID NO:1 or 3 is one which is sufficiently complementary to thenucleotide sequence shown in SEQ ID NO:1 or 3 or to the homologoussequence such that it can hybridize to the nucleotide sequence shown inSEQ ID NO:1 or 3 or to the homologous sequence, thereby forming a stableduplex.

In still another preferred embodiment, an isolated nucleic acid moleculeof the invention comprises a nucleotide sequence which is at least about50%, preferably at least about 60%, more preferably at least about 70%,yet more preferably at least about 80%, still more preferably at leastabout 90%, and most preferably at least about 95% or more homologous tothe nucleotide sequence shown in SEQ ID NO:1 or 3 or a portion of thisnucleotide sequence. In an additional preferred embodiment, an isolatednucleic acid molecule of the invention comprises a nucleotide sequencewhich hybridizes, i.e., hybridizes under stringent conditions, to thenucleotide sequence shown in SEQ ID NO:1 or 3 or to a nucleotidesequence which is at least about 50%, preferably at least about 60%,more preferably at least about 70%, yet more preferably at least about80%, still more preferably at least about 90%, and most preferably atleast about 95% or more homologous to the nucleotide sequence shown inSEQ ID NO:1 or 3.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the coding region of SEQ ID NO:1 or 3 or the coding region ofa nucleotide sequence which is at least about 50%, preferably at leastabout 60%, more preferably at least about 70%, yet more preferably atleast about 80%, still more preferably at least about 90%, and mostpreferably at least about 95% or more homologous to the nucleotidesequence shown in SEQ ID NO:1 or 3, for example a fragment which can beused as a probe or primer or a fragment encoding a biologically activeportion of CXCR4 and/or c-Kit. The nucleotide sequence determined fromthe cloning of the CXCR4 and/or c-Kit gene from a mouse or human allowsfor the generation of probes and primers designed for use in identifyingand/or cloning other CXCR4 and/or c-Kit family members, as well as CXCR4and/or c-Kit homologues in other cell types, i.e. from other tissues, aswell as CXCR4 and/or c-Kit homologues from other mammals such as rats ormonkeys. The probe/primer typically comprises substantially purifiedoligonucleotide. The oligonucleotide typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12, preferably at least about 25, more preferably about 40,50 or 75 consecutive nucleotides of SEQ ID NO:1 or SEQ ID NO:3 sense, ananti-sense sequence of SEQ ID NO:1 or SEQ ID NO:3, or naturallyoccurring mutants thereof. Primers based on the nucleotide sequence inSEQ ID NO:1 or SEQ ID NO:3 can be used in PCR reactions to clone CXCR4and/or c-Kit homologues.

Probes based on the CXCR4 and/or c-Kit nucleotide sequences can be usedto detect transcripts or genomic sequences encoding the same orhomologous proteins. In preferred embodiments, the probe furthercomprises a label group attached thereto, i.e. the label group can be aradioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.Such probes can be used as a part of a diagnostic test kit foridentifying cells or tissue (e.g., lung cells or lung tumor cells) whichexpress or misexpress a CXCR4 and/or c-Kit protein, such as by measuringa level of a CXCR4 and/or c-Kit encoding nucleic acid in a sample ofcells (e.g., lung cells) from a subject, i.e., detecting CXCR4 and/orc-Kit mRNA levels or determining whether a genomic CXCR4 and/or c-Kitgene has been mutated or deleted.

In one embodiment, the nucleic acid molecule of the invention encodes aprotein or portion thereof which includes an amino acid sequence whichis sufficiently homologous to an amino acid sequence of SEQ ID NO:2 or 4such that the protein or portion thereof maintains one or more of thefollowing biological activities: 1) it can modulate growth of SCLCcells; 2) it can modulate proliferation of SCLC cells; 3) it canmodulate movement of SCLC cells; 4) it can modulate motility of SCLCcells; 5) it can modulate adhesion of SCLC cells; 6) it can modulatecellular shape and/or morphological change of SCLC cells; 7) it canmodulate metastasis of SCLC cells; 8) it can modulate CXCR4 activity; 9)it can modulate CXCR4 and c-Kit activity; 10) it can modulate CXCR4binding to SDF-1α; 11) it can modulate c-Kit binding to SCF; and/or 12)it can modulate PI3-K activity.

As used herein, the language “sufficiently homologous” refers toproteins or portions thereof which have amino acid sequences whichinclude a minimum number of identical or equivalent (i.e., an amino acidresidue which has a similar side chain as an amino acid residue in SEQID NO:2 or 4) amino acid residues to an amino acid sequence of SEQ IDNO:2 or 4 such that the protein or portion thereof maintains one or moreof the following biological activities: 1) it can modulate growth ofSCLC cells; 2) it can modulate proliferation of SCLC cells; 3) it canmodulate movement of SCLC cells; 4) it can modulate motility of SCLCcells; 5) it can modulate adhesion of SCLC cells; 6) it can modulatecellular shape and/or morphological change of SCLC cells; 7) it canmodulate metastasis of SCLC cells; 8) it can modulate CXCR4 activity; 9)it can modulate CXCR4 and c-Kit activity; 10) it can modulate CXCR4binding to SDF-1α; 11) it can modulate c-Kit binding to SCF; and/or 12)it can modulate PI3-K activity.

In another embodiment, the protein is at least about 50%, preferably atleast about 60%, more preferably at least about 70%, yet more preferablyat least about 80%, still more preferably at least about 90%, and mostpreferably at least about 95% or more homologous to the entire aminoacid sequence of SEQ ID NO:2 or 4.

Portions of proteins encoded by the CXCR4 and/or c-Kit nucleic acidmolecule of the invention are preferably biologically active portions ofthe CXCR4 and/or c-Kit protein. As used herein, the term “biologicallyactive portion of CXCR4 and/or c-Kit” is intended to include a portion,i.e., a domain/motif, of CXCR4 and/or c-Kit that has one or more of thefollowing activities: 1) it can modulate growth of SCLC cells; 2) it canmodulate proliferation of SCLC cells; 3) it can modulate movement ofSCLC cells; 4) it can modulate motility of SCLC cells; 5) it canmodulate adhesion of SCLC cells; 6) it can modulate cellular shapeand/or morphological change of SCLC cells; 7) it can modulate metastasisof SCLC cells; 8) it can modulate CXCR4 activity; 9) it can modulateCXCR4 and c-Kit activity; 10) it can modulate CXCR4 binding to SDF-1α;11) it can modulate c-Kit binding to SCF; and/or 12) it can modulatePI3-K activity.

Standard binding assays, i.e., immunoprecipitations and yeast two-hybridassays, as described herein, can be performed to determine the abilityof a CXCR4 or c-Kit protein or a biologically active portion thereof tointeract with (i.e., bind to) SDF-1α or SCF, respectively.

In one embodiment, the biologically active portion of CXCR4 and/or c-Kitcomprises at least one domain or motif. In one embodiment, thebiologically active portion of the protein which includes the domain ormotif can modulate proliferation or metastasis of SCLC cells or canmodulate PI3-K signaling. These domains are described herein. Additionalnucleic acid fragments encoding biologically active portions of CXCR4and/or c-Kit can be prepared by isolating a portion of SEQ ID NO:1 or 3or a homologous nucleotide sequence, expressing the encoded portion ofCXCR4 and/or c-Kit protein or peptide (i.e., by recombinant expressionin vitro) and assessing the activity of the encoded portion of CXCR4and/or c-Kit protein or peptide.

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence shown in SEQ ID NO:1 or 3 (and portionsthereof) due to degeneracy of the genetic code and thus encode the sameCXCR4 and/or c-Kit protein as that encoded by the nucleotide sequenceshown in SEQ ID NO:1 or 3. In another embodiment, an isolated nucleicacid molecule of the invention has a nucleotide sequence encoding aprotein having an amino acid sequence shown in SEQ ID NO:2 or 4 or aprotein having an amino acid sequence which is at least about 50%,preferably at least about 60%, more preferably at least about 70%, yetmore preferably at least about 80%, still more preferably at least about90%, and most preferably at least about 95% or more homologous to theamino acid sequence of SEQ ID NO:2 or 4.

In addition to the human CXCR4 and c-Kit nucleotide sequences shown inSEQ ID NO:1 and 3, respectively, it will be appreciated by those skilledin the art that DNA sequence polymorphisms that lead to changes in theamino acid sequences of CXCR4 and/or c-Kit may exist within a population(i.e., a mammalian population, i.e., a human population). Such geneticpolymorphism in the CXCR4 or c-Kit gene may exist among individualswithin a population due to natural allelic variation. As used herein,the terms “gene” and “recombinant gene” refer to nucleic acid moleculescomprising an open reading frame encoding a CXCR4 or c-Kit protein,preferably a mammalian, i.e., human, CXCR4 or c-Kit protein. Suchnatural allelic variations can typically result in 1-5% variance in thenucleotide sequence of the CXCR4 or c-Kit gene. Any and all suchnucleotide variations and resulting amino acid polymorphisms in CXCR4and/or c-Kit that are the result of natural allelic variation and thatdo not alter the functional activity of CXCR4 and/or c-Kit are intendedto be within the scope of the methods of the invention. Moreover,nucleic acid molecules encoding CXCR4 and/or c-Kit proteins from otherspecies, and thus which have a nucleotide sequence which differs fromthe human or mouse sequences of SEQ ID NO:1 or 3 are intended to bewithin the scope of the invention. Nucleic acid molecules correspondingto natural allelic variants and homologues of the mouse or human CXCR4and/or c-Kit cDNAs of the invention can be isolated based on theirhomology to the human CXCR4 and/or c-Kit nucleic acid sequencesdisclosed herein using the human cDNA, or a portion thereof, as ahybridization probe according to standard hybridization techniques understringent hybridization conditions (as described herein).

Moreover, nucleic acid molecules encoding other CXCR4 and/or c-Kitfamily members and thus which have a nucleotide sequence which differsfrom the CXCR4 and/or c-Kit sequences of SEQ ID NO:1 or 3 are intendedto be within the scope of the invention. For example, the use ofalternately-spliced isoforms of CXCR4 and/or c-Kit, other CXCR4 and/orc-Kit family members, or CXCR4 and/or c-Kit members from other species,and thus which have a nucleotide sequence which differs from the CXCR4and c-Kit sequences of SEQ ID NO:1 and 3 are intended to be within thescope of the invention. For example, rat or monkey CXCR4 or c-Kit cDNAcan be identified based on the nucleotide sequence of a human CXCR4 orc-Kit, respectively.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 15 nucleotides in length and hybridizes understringent conditions to the nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:1 or 3 or a nucleotide sequence whichis about 60%, preferably at least about 70%, more preferably at leastabout 80%, still more preferably at least about 90%, and most preferablyat least about 95% or more homologous to the nucleotide sequence of SEQID NO:1 or 3. In other embodiments, the nucleic acid is at least 30, 50,100, 250 or 500 nucleotides in length. As used herein, the term“hybridizes under stringent conditions” is intended to describeconditions for hybridization and washing under which nucleotidesequences at least 60% homologous to each other typically remainhybridized to each other. Preferably, the conditions are such thatsequences at least about 65%, more preferably at least about 70%, andeven more preferably at least about 75% or more homologous to each othertypically remain hybridized to each other. Such stringent conditions areknown to those skilled in the art and can be found in Current Protocolsin Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Apreferred, non-limiting example of stringent hybridization conditionsare hybridization in 6× sodium chloride/sodium citrate (SSC) at about45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C.Preferably, an isolated nucleic acid molecule of the invention thathybridizes under stringent conditions to the sequence of SEQ ID NO:1 orSEQ ID NO:3 corresponds to a naturally-occurring nucleic acid molecule.As used herein, a “naturally-occurring” nucleic acid molecule refers toan RNA or DNA molecule having a nucleotide sequence that occurs innature (i.e., encodes a natural protein). In one embodiment, the nucleicacid encodes a natural human CXCR4 or c-Kit.

In addition to naturally-occurring allelic variants of the CXCR4 orc-Kit sequence that may exist in the population, the skilled artisanwill further appreciate that changes can be introduced by mutation intothe nucleotide sequence of SEQ ID NO:1 or 3, thereby leading to changesin the amino acid sequence of the encoded CXCR4 or c-Kit protein,without altering the functional ability of the CXCR4 or c-Kit protein.For example, nucleotide substitutions leading to amino acidsubstitutions at “non-essential” amino acid residues can be made in thesequence of SEQ ID NO:1 or 3. A non-essential” amino acid residue is aresidue that can be altered from the wild-type sequence of CXCR4 orc-Kit (i.e., the sequence of SEQ ID NO:2 or 4, respectively) withoutaltering the activity of CXCR4 or c-Kit, whereas an “essential” aminoacid residue is required for CXCR4 or c-Kit activity. For example, aminoacid residues involved in the interaction of CXCR4 or c-Kit to bindingpartners or target molecules (e.g., SDF-1α or SCF, respectively) aremost likely essential residues of CXCR4 or c-Kit. Other amino acidresidues, however, (i.e., those that are not conserved or onlysemi-conserved between mouse and human) may not be essential foractivity and thus are likely to be amenable to alteration withoutaltering CXCR4 or c-Kit activity. Furthermore, amino acid residues thatare essential for CXCR4 or c-Kit functions related to SCLC proliferationor metastasis, but not essential for CXCR4 or c-Kit functions related toHIV infection, are likely to be amenable to alteration.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding CXCR4 or c-Kit proteins that contain changes in aminoacid residues that are not essential for CXCR4 or c-Kit activity. SuchCXCR4 and c-Kit proteins differ in amino acid sequence from SEQ ID NOs:2and 4 yet retain at least one of the CXCR4 or c-Kit activities describedherein. In one embodiment, the isolated nucleic acid molecule comprisesa nucleotide sequence encoding a protein, wherein the protein comprisesan amino acid sequence at least about 60% homologous to the amino acidsequence of SEQ ID NO:2 or 4 and is capable of modulating proliferationor metastasis. Preferably, the protein encoded by the nucleic acidmolecule is at least about 70% homologous, preferably at least about80-85% homologous, still more preferably at least about 90%, and mostpreferably at least about 95% homologous to the amino acid sequence ofSEQ ID NO:2 or 4.

“Sequence identity or homology”, as used herein, refers to the sequencesimilarity between two polypeptide molecules or between two nucleic acidmolecules. When a position in both of the two compared sequences isoccupied by the same base or amino acid monomer subunit, i.e., if aposition in each of two DNA molecules is occupied by adenine, then themolecules are homologous or sequence identical at that position. Thepercent of homology or sequence identity between two sequences is afunction of the number of matching or homologous identical positionsshared by the two sequences divided by the number of positions compared×100. For example, if 6 of 10, of the positions in two sequences are thesame then the two sequences are 60% homologous or have 60% sequenceidentity. By way of example, the DNA sequences ATTGCC and TATGGC share50% homology or sequence identity. Generally, a comparison is made whentwo sequences are aligned to give maximum homology. Unless otherwisespecified “loop out regions”, i.e., those arising from, from deletionsor insertions in one of the sequences are counted as mismatches.

The comparison of sequences and determination of percent homologybetween two sequences can be accomplished using a mathematicalalgorithm. Preferably, the alignment can be performed using the ClustalMethod. Multiple alignment parameters include GAP Penalty=10, Gap LengthPenalty=10. For DNA alignments, the pairwise alignment parameters can beHtuple=2, Gap penalty=5, Window=4, and Diagonal saved=4. For proteinalignments, the pairwise alignment parameters can be Ktuple=1, Gappenalty=3, Window=5, and Diagonals Saved=5.

In a preferred embodiment, the percent identity between two amino acidsequences is determined using the Needleman and Wunsch (J. Mol. Biol.(48):444-453 (1970)) algorithm which has been incorporated into the GAPprogram in the GCG software package (available online), using either aBlossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12,10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yetanother preferred embodiment, the percent identity between twonucleotide sequences is determined using the GAP program in the GCGsoftware package (available online), using a NWSgapdna.CMP matrix and agap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4,5, or 6. In another embodiment, the percent identity between two aminoacid or nucleotide sequences is determined using the algorithm of E.Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has beenincorporated into the ALIGN program (version 2.0) (available online),using a PAM120 weight residue table, a gap length penalty of 12 and agap penalty of 4.

An isolated nucleic acid molecule encoding a CXCR4 protein homologous tothe protein of SEQ ID NO:2 or 4 can be created by introducing one ormore nucleotide substitutions, additions or deletions into thenucleotide sequence of SEQ ID NO:1 or 3 or a homologous nucleotidesequence such that one or more amino acid substitutions, additions ordeletions are introduced into the encoded protein. Mutations can beintroduced into SEQ ID NO:1 or 3 or the homologous nucleotide sequenceby standard techniques, such as site-directed mutagenesis andPCR-mediated mutagenesis. Preferably, conservative amino acidsubstitutions are made at one or more predicted non-essential amino acidresidues. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (i.e., lysine, arginine, histidine), acidic sidechains (i.e., aspartic acid, glutamic acid), uncharged polar side chains(i.e., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (i.e., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (i.e., threonine, valine, isoleucine) andaromatic side chains (i.e., tyrosine, phenylalanine, tryptophan,histidine). Thus, a predicted nonessential amino acid residue in CXCR4or c-Kit is preferably replaced with another amino acid residue from thesame side chain family. Alternatively, in another embodiment, mutationscan be introduced randomly along all or part of a CXCR4 or c-Kit codingsequence, such as by saturation mutagenesis, and the resultant mutantscan be screened for a CXCR4 or c-Kit activity described herein toidentify mutants that retain CXCR4 or c-Kit activity. Followingmutagenesis of SEQ ID NO:1 or 3, the encoded protein can be expressedrecombinantly (as described herein) and the activity of the protein canbe determined using, for example, assays described herein.

In addition to the nucleic acid molecules encoding CXCR4 and c-Kitproteins described above, another aspect of the invention pertains toisolated nucleic acid molecules which are antisense thereto. An“antisense” nucleic acid comprises a nucleotide sequence which iscomplementary to a “sense” nucleic acid encoding a protein, i.e.,complementary to the coding strand of a double-stranded cDNA molecule orcomplementary to an mRNA sequence. Accordingly, an antisense nucleicacid can hydrogen bond to a sense nucleic acid. The antisense nucleicacid can be complementary to an entire CXCR4 or c-Kit coding strand, orto only a portion thereof. In one embodiment, an antisense nucleic acidmolecule is antsense to a “coding region” of the coding strand of anucleotide sequence encoding CXCR4 or c-Kit. The term “coding region”refers to the region of the nucleotide sequence comprising codons whichare translated into amino acid residues (i.e., the entire coding regionof SEQ ID NO:1 comprises nucleotides 89-1144, and the entire codingregion of SEQ ID NO:4 comprises nucleotides 22 to 2949). In anotherembodiment, the antisense nucleic acid molecule is antisense to a“noncoding region” of the coding strand of a nucleotide sequenceencoding CXCR4 or c-Kit. The term “noncoding region” refers to 5′ and 3′sequences which flank the coding region that are not translated intoamino acids (i.e., also referred to as 5′ and 3′ untranslated regions).

Given the coding strand sequences encoding CXCR4 and c-Kit disclosedherein, antisense nucleic acids of the invention can be designedaccording to the rules of Watson and Crick base pairing. The antisensenucleic acid molecule can be complementary to the entire coding regionof CXCR4 or c-Kit mRNA, but more preferably is an oligonucleotide whichis antisense to only a portion of the coding or noncoding region ofCXCR4 or c-Kit mRNA. For example, the antisense oligonucleotide can becomplementary to the region surrounding the translation start site ofCXCR4 or c-Kit mRNA. An antisense oligonucleotide can be, for example,about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. Anantisense nucleic acid of the invention can be constructed usingchemical synthesis and enzymatic ligation reactions using proceduresknown in the art. For example, an antisense nucleic acid (i.e., anantisense oligonucleotide) can be chemically synthesized using naturallyoccurring nucleotides or variously modified nucleotides designed toincrease the biological stability of the molecules or to increase thephysical stability of the duplex formed between the antisense and sensenucleic acids, i.e., phosphorothioate derivatives and acridinesubstituted nucleotides can be used. Examples of modified nucleotideswhich can be used to generate the antisense nucleic acid include5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6 isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine,pseudouracil, queosine, 2-thiocytosine, 5methyl-2-thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acidmethylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.Alternatively, the antsense nucleic acid can be produced biologicallyusing an expression vector into which a nucleic acid has been subclonedin an antisense orientation (i.e., RNA transcribed from the insertednucleic acid will be of an antisense orientation to a target nucleicacid of interest, described further in the following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a CXCR4 orc-Kit protein to thereby inhibit expression of the protein, i.e., byinhibiting transcription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. An example of a route of administration of anantisense nucleic acid molecule of the invention includes directinjection at a tissue site. Alternatively, an antisense nucleic acidmolecule can be modified to target selected cells and then administeredsystemically. For example, for systemic administration, an antisensemolecule can be modified such that it specifically binds to a receptoror an antigen expressed on a selected cell surface, i.e., by linking theantisense nucleic acid molecule to a peptide or an antibody which bindsto a cell surface receptor or antigen. The antisense nucleic acidmolecule can also be delivered to cells using the vectors describedherein. To achieve sufficient intracellular concentrations of theantisense molecules, vector constructs in which the antisense nucleicacid molecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al. (1987) Nucleic Acids Res. 15:6625-6641). Theantisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

In still another embodiment, an antisense nucleic acid of the inventionis a ribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity which are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes (i.e., hammerhead ribozymes (described in Haseloff and Gerlach(1988) Nature 334:585-591)) can be used to catalytically cleave CXCR4 orc-Kit mRNA transcripts to thereby inhibit translation of CXCR4 or c-KitmRNA A ribozyme having specificity for a CXCR4 or c-Kit encoding nucleicacid can be designed based upon the nucleotide sequence of a CXCR4 orc-Kit cDNA disclosed herein (i.e., SEQ ID NO:1 or 3). For example, aderivative of a Tetrahymena L-19 IVS RNA can be constructed in which thenucleotide sequence of the active site is complementary to thenucleotide sequence to be cleaved in a CXCR4 or c-Kit encoding mRNA.See, i.e., Cech et al. U.S. Pat. No. 4,987,071 and Cech et al. U.S. Pat.No. 5,116,742. Alternatively, CXCR4 mRNA can be used to select acatalytic RNA having a specific ribonuclease activity from a pool of RNAmolecules. See, i.e., Bartel, D. and Szostak, J. W. (1993) Science261:1411-1418.

Alternatively, CXCR4 or c-Kit gene expression can be inhibited bytargeting nucleotide sequences complementary to the regulatory region ofthe CXCR4 or c-Kit (i.e., the CXCR4 or c-Kit promoter and/or enhancers)to form triple helical structures that prevent transcription of theCXCR4 or c-Kit gene in target cells. See generally, Helene, C. (1991)Anticancer Drug Des. 6(6):569-84; Helene, C. et al. (1992) Ann. N.Y.Acad. Sci. 660:27-36; and Maher, L. J. (1992) Bioassays 14(12):807-15.

II. Isolated CXCR4 and c-Kit Proteins and Anti-CXCR4 and c-KitAntibodies

Another aspect of the invention pertains to the use of isolated CXCR4 orc-Kit proteins, and biologically active portions thereof, as well aspeptide fragments suitable for use as immunogens to raise anti-CXCR4 andc-Kit antibodies. An “isolated” or “purified” protein or biologicallyactive portion thereof is substantially free of cellular material whenproduced by recombinant DNA techniques, or chemical precursors or otherchemicals when chemically synthesized. The language “substantially freeof cellular material” includes preparations of CXCR4 or c-Kit protein inwhich the protein is separated from cellular components of the cells inwhich it is naturally or recombinantly produced. In one embodiment, thelanguage “substantially free of cellular material includes preparationsof CXCR4 or c-Kit protein having less than about 30% (by dry weight) ofnon-CXCR4 or c-Kit protein (also referred to herein as a “contaminatingprotein”), more preferably less than about 20% of non-CXCR4 or c-Kitprotein, still more preferably less than about 10% of non-CXCR4 or c-Kitprotein, and most preferably less than about 5% non-CXCR4 or c-Kitprotein. When the CXCR4 or c-Kit protein or biologically active portionthereof is recombinantly produced, it is also preferably substantiallyfree of culture medium, i.e., culture medium represents less than about20%, more preferably less than about 10%, and most preferably less thanabout 5% of the volume of the protein preparation. The language“substantially free of chemical precursors or other chemicals” includespreparations of CXCR4 or c-Kit protein in which the protein is separatedfrom chemical precursors or other chemicals which are involved in thesynthesis of the protein. In one embodiment, the language “substantiallyfree of chemical precursors or other chemicals” includes preparations ofCXCR4 or c-Kit protein having less than about 30% (by dry weight) ofchemical precursors or non-CXCR4 or c-Kit chemicals, more preferablyless than about 20% chemical precursors or non-CXCR4 or c-Kit chemicals,still more preferably less than about 10% chemical precursors ornon-CXCR4 or c-Kit chemicals, and most preferably less than about 5%chemical precursors or non-CXCR4 or c-Kit chemicals. In preferredembodiments, isolated proteins or biologically active portions thereoflack contaminating proteins from the same animal from which the CXCR4 orc-Kit protein is derived. Typically, such proteins are produced byrecombinant expression of, for example, a human CXCR4 or c-Kit proteinin a nonhuman cell.

An isolated CXCR4 protein or a portion thereof of the invention has oneor more of the following biological activities: 1) it can modulategrowth of SCLC cells; 2) it can modulate proliferation of SCLC cells; 3)it can modulate movement of SCLC cells; 4) it can modulate motility ofSCLC cells; 5) it can modulate adhesion of SCLC cells; 6) it canmodulate cellular shape and/or morphological change of SCLC cells; 7) itcan modulate metastasis of SCLC cells; 8) it can modulate CXCR4activity; 9) it can modulate CXCR4 and c-Kit activity; 10) it canmodulate CXCR4 binding to SDF-1α; 11) it can modulate c-Kit binding toSCF; and/or 12) it can modulate PI3-K activity.

In preferred embodiments, the protein or portion thereof comprises anamino acid sequence which is sufficiently homologous to an amino acidsequence of SEQ ID NO:2 or 4 such that the protein or portion thereofmaintains the ability to modulate any of the above-described CXCR4 orc-Kit activities. The portion of the protein is preferably abiologically active portion as described herein. In another preferredembodiment, the CXCR4 or c-Kit protein has an amino acid sequence shownin SEQ ID NO:2 or 4, or an amino acid sequence which is at least about50%, preferably at least about 60%, more preferably at least about 70%,yet more preferably at least about 80%, still more preferably at leastabout 90%, and most preferably at least about 95% or more homologous tothe amino acid sequence shown in SEQ ID NO:2 or 4. In yet anotherpreferred embodiment, the CXCR4 or c-Kit protein has an amino acidsequence which is encoded by a nucleotide sequence which hybridizes,i.e., hybridizes under stringent conditions, to the nucleotide sequenceof SEQ ID NO:1 or 3 or a nucleotide sequence which is at least about50%, preferably at least about 60%, more preferably at least about 70%,yet more preferably at least about 80%, still more preferably at leastabout 90%, and most preferably at least about 95% or more homologous tothe nucleotide sequence shown in SEQ ID NO:1 or 3. The preferred CXCR4or c-Kit proteins used in the methods of the present invention alsopreferably possess at least one of the CXCR4 or c-Kit biologicalactivities described herein. For example, a preferred CXCR4 or c-Kitprotein used in the methods of the present invention includes an aminoacid sequence encoded by a nucleotide sequence which hybridizes, i.e.,hybridizes under stringent conditions, to the nucleotide sequence of SEQID NO:1 or 3 and which can modulate any of the above-described CXCR4 orc-Kit activities.

In other embodiments, the CXCR4 or c-Kit protein is substantiallyhomologous to the amino acid sequence of SEQ ID NO:2 or 4 and retainsthe functional activity of the protein of SEQ ID NO:2 or 4, yet differsin amino acid sequence due to natural allelic variation or mutagenesis,as described in detail in subsection I above. Accordingly, in anotherembodiment, the CXCR4 or c-Kit protein is a protein which comprises anamino acid sequence which is at least about 50%, preferably at leastabout 60%, more preferably at least about 70%, yet more preferably atleast about 80%, still more preferably at least about 90%, and mostpreferably at least about 95% or more homologous to the amino acidsequence of SEQ ID NO:2 or 4.

Biologically active portions of the CXCR4 or c-Kit protein includepeptides comprising amino acid sequences derived from the amino acidsequence of the CXCR4 or c-Kit protein, i.e., the amino acid sequenceshown in SEQ ID NO:2 or 4 or the amino acid sequence of a proteinhomologous to the CXCR4 or c-Kit protein, which include fewer aminoacids than the full length CXCR4 or c-Kit protein or the full lengthprotein which is homologous to the CXCR4 or c-Kit protein, and exhibitat least one activity of the CXCR4 or c-Kit protein. Typically,biologically active portions (peptides, i.e., peptides which are, forexample, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or moreamino acids in length) comprise a domain or motif, with at least oneactivity of the CXCR4 or c-Kit protein. Moreover, other biologicallyactive portions, in which other regions of the protein are deleted, canbe prepared by recombinant techniques and evaluated for one or more ofthe activities described herein. Preferably, the biologically activeportions of the CXCR4 or c-Kit protein include one or more selecteddomains/motifs or portions thereof having biological activity.

CXCR4 or c-Kit proteins are preferably produced by recombinant DNAtechniques. For example, a nucleic acid molecule encoding the protein iscloned into an expression vector (as described above), the expressionvector is introduced into a host cell (as described above) and the CXCR4or c-Kit protein is expressed in the host cell. The CXCR4 or c-Kitprotein can then be isolated from the cells by an appropriatepurification scheme using standard protein purification techniques.Alternative to recombinant expression, a CXCR4 or c-Kit protein,polypeptide, or peptide can be synthesized chemically using standardpeptide synthesis techniques. Moreover, native CXCR4 or c-Kit proteincan be isolated from cells (i.e., lung cells or lung tumor cells), forexample using an anti-CXCR4 or c-Kit antibody (described further below).

The invention also provides for the use of CXCR4 or c-Kit chimeric orfusion proteins. As used herein, a CXCR4 or c-Kit “chimeric protein” or“fusion protein” comprises a CXCR4 or c-Kit polypeptide operativelylinked to a non-CXCR4 or c-Kit polypeptide. A “CXCR4 or c-Kitpolypeptide” refers to a polypeptide having an amino acid sequencecorresponding to CXCR4 or c-Kit, respectively, whereas a “non-CXCR4 orc-Kit polypeptide” refers to a polypeptide having an amino acid sequencecorresponding to a protein which is not substantially homologous to theCXCR4 or c-Kit protein, i.e., a protein which is different from theCXCR4 or c-Kit protein and which is derived from the same or a differentorganism. Within the fusion protein, the term “operatively linked” isintended to indicate that the CXCR4 or c-Kit polypeptide and thenon-CXCR4 or c-Kit polypeptide are fused in-frame to each other. Thenon-CXCR4 or c-Kit polypeptide can be fused to the N-terminus orC-terminus of the CXCR4 or c-Kit polypeptide. For example, in oneembodiment the fusion protein is a GST-CXCR4 or c-Kit fusion protein inwhich the CXCR4 or c-Kit sequences are fused to the C-terminus of theGST sequences. Such fusion proteins can facilitate the purification ofrecombinant CXCR4 or c-Kit. In another embodiment, the fusion protein isa CXCR4 or c-Kit protein containing a heterologous signal sequence atits N-terminus. In certain host cells (i.e., mammalian host cells),expression and/or secretion of CXCR4 or c-Kit can be increased throughuse of a heterologous signal sequence.

Preferably, a CXCR4 or c-Kit chimeric or fusion protein of the inventionis produced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and enzymatic ligation. Inanother embodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed and reamplified to generatea chimeric gene sequence (see, for example, Current Protocols inMolecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (i.e., a GST polypeptide). A CXCR4 orc-Kit encoding nucleic acid can be cloned into such an expression vectorsuch that the fusion moiety is linked in-frame to the CXCR4 or c-Kitprotein.

The present invention also pertains to the use of homologues of theCXCR4 or c-Kit proteins which function as either a CXCR4 or c-Kitagonist (mimetic) or a CXCR4 or c-Kit antagonist. In a preferredembodiment, the CXCR4 or c-Kit agonists and antagonists stimulate orinhibit, respectively, a subset of the biological activities of thenaturally occurring form of the CXCR4 or c-Kit protein. Thus, specificbiological effects can be elicited by treatment with a homologue oflimited function. In one embodiment, treatment of a subject with ahomologue having a subset of the biological activities of the naturallyoccurring form of the protein has fewer side effects in a subjectrelative to treatment with the naturally occurring form of the CXCR4 orc-Kit protein.

Homologues of the CXCR4 or c-Kit protein can be generated bymutagenesis, i.e., discrete point mutation or truncation of the CXCR4 orc-Kit protein. As used herein, the term “homologue” refers to a variantform of the CXCR4 or c-Kit protein which acts as an agonist orantagonist of the activity of the CXCR4 or c-Kit protein. An agonist ofthe CXCR4 or c-Kit protein can retain substantially the same, or asubset, of the biological activities of the CXCR4 or c-Kit protein. Anantagonist of the CXCR4 or c-Kit protein can inhibit one or more of theactivities of the naturally occurring form of the CXCR4 or c-Kitprotein, by, for example, competitively binding to a downstream orupstream member of the CXCR4 or c-Kit cascade which includes the CXCR4or c-Kit protein. Thus, the mammalian CXCR4 or c-Kit protein andhomologues thereof of the present invention can be, for example, eitherpositive or negative regulators of SCLC proliferation or metastasis.

In an alternative embodiment, homologues of the CXCR4 or c-Kit proteincan be identified by screening combinatorial libraries of mutants, i.e.,truncation mutants, of the CXCR4 or c-Kit protein for CXCR4 or c-Kitprotein agonist or antagonist activity. In one embodiment, a variegatedlibrary of CXCR4 or c-Kit variants is generated by combinatorialmutagenesis at the nucleic acid level and is encoded by a variegatedgene library. A variegated library of CXCR4 or c-Kit variants can beproduced by, for example, enzymatically lighting a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential CXCR4 or c-Kit sequences is expressible as individualpolypeptides, or alternatively, as a set of larger fusion proteins(i.e., for phage display) containing the set of CXCR4 or c-Kit sequencestherein. There are a variety of methods which can be used to producelibraries of potential CXCR4 or c-Kit homologues from a degenerateoligonucleotide sequence. Chemical synthesis of a degenerate genesequence can be performed in an automatic DNA synthesizer, and thesynthetic gene then ligated into an appropriate expression vector. Useof a degenerate set of genes allows for the provision, in one mixture,of all of the sequences encoding the desired set of potential CXCR4 orc-Kit sequences. Methods for synthesizing degenerate oligonucleotidesare known in the art (see, i.e., Narang, S. A. (1983) Tetrahedron 39:3;Itakura et a. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984)Science 198:1056; Ike et al. (1983) Nucleic Acids Res. 11:477.

In addition, libraries of fragments of the CXCR4 or c-Kit protein codingcan be used to generate a variegated population of CXCR4 or c-Kitfragments for screening and subsequent selection of homologues of aCXCR4 or c-Kit protein. In one embodiment, a library of coding sequencefragments can be generated by treating a double stranded PCR fragment ofa CXCR4 or c-Kit coding sequence with a nuclease under conditionswherein nicking occurs only about once per molecule, denaturing thedouble stranded DNA, renaturing the DNA to form double stranded DNAwhich can include sense/antisense pairs from different nicked products,removing single stranded portions from reformed duplexes by treatmentwith S1 nuclease, and ligating the resulting fragment library into anexpression vector. By this method, an expression library can be derivedwhich encodes N-terminal, C-terminal and internal fragments of varioussizes of the CXCR4 or c-Kit protein.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of CXCR4 or c-Kit homologues.The most widely used techniques, which are amenable to high through-putanalysis, for screening large gene libraries typically include cloningthe gene library into replicable expression vectors, transformingappropriate cells with the resulting library of vectors, and expressingthe combinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a newtechnique which enhances the frequency of functional mutants in thelibraries, can be used in combination with the screening assays toidentify CXCR4 homologues (Arkin and Youvan (1992) Proc. Natl. Acad.Sci. USA 89:7811-7815; Delagrave et al. (1993) Protein Eng.6(3):327-331).

An isolated CXCR4 or c-Kit protein, or a portion or fragment thereof,can be used as an immunogen to generate antibodies that bind CXCR4 orc-Kit using standard techniques for polyclonal and monoclonal antibodypreparation. The full-length CXCR4 or c-Kit protein can be used or,alternatively, the invention provides antigenic peptide fragments ofCXCR4 or c-Kit for use as immunogens. The antigenic peptide of CXCR4 orc-Kit comprises at least 8 amino acid residues of the amino acidsequence shown in SEQ ID NO:2 or 4 or a homologous amino acid sequenceas described herein and encompasses an epitope of CXCR4 or c-Kit suchthat an antibody raised against the peptide forms a specific immunecomplex with CXCR4 or c-Kit. Preferably, the antigenic peptide comprisesat least 10 amino acid residues, more preferably at least 15 amino acidresidues, even more preferably at least 20 amino acid residues, and mostpreferably at least 30 amino acid residues. Preferred epitopesencompassed by the antigenic peptide are regions of CXCR4 or c-Kit thatare located on the surface of the protein, i.e., hydrophilic regions.

A CXCR4 or c-Kit immunogen typically is used to prepare antibodies byimmunizing a suitable subject, (i.e., rabbit, goat, mouse or othermammal) with the immunogen. An appropriate immunogenic preparation cancontain, for example, recombinantly expressed CXCR4 or c-Kit protein ora chemically synthesized CXCR4 or c-Kit peptide. The preparation canfurther include an adjuvant, such as Freund's complete or incompleteadjuvant, or similar immunostimulatory agent. Immunization of a suitablesubject with an immunogenic CXCR4 or c-Kit preparation induces apolyclonal anti-CXCR4 or c-Kit antibody response.

Accordingly, another aspect of the invention pertains to the use ofanti-CXCR4 or c-Kit antibodies. The term “antibody” as used hereinrefers to immunoglobulin molecules and immunologically active portionsof immunoglobulin molecules, i.e., molecules that contain an antigenbinding site which specifically binds (immunoreacts with) an antigen,such as CXCR4 or c-Kit. Examples of immunologically active portions ofimmunoglobulin molecules include F(ab) and F(ab′)2 fragments which canbe generated by treating the antibody with an enzyme such as pepsin. Theinvention provides polyclonal and monoclonal antibodies that bind CXCR4or c-Kit. The term “monoclonal antibody” or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one species of an antigen binding sitecapable of immunoreacting with a particular epitope of CXCR4 or c-Kit. Amonoclonal antibody composition thus typically displays a single bindingaffinity for a particular CXCR4 or c-Kit protein with which itimmunoreacts.

Polyclonal anti-CXCR4 or c-Kit antibodies can be prepared as describedabove by immunizing a suitable subject with a CXCR4 or c-Kit immunogen.The anti-CXCR4 or c-Kit antibody titer in the immunized subject can bemonitored over time by standard techniques, such as with an enzymelinked immunosorbent assay (ELISA) using immobilized CXCR4 or c-Kit. Ifdesired, the antibody molecules directed against CXCR4 or c-Kit can beisolated from the mammal (i.e., from the blood) and further purified bywell known techniques, such as protein A chromatography to obtain theIgG fraction. At an appropriate time after immunization, i.e., when theanti-CXCR4 or c-Kit antibody titers are highest, antibody-producingcells can be obtained from the subject and used to prepare monoclonalantibodies by standard techniques, such as the hybridoma techniqueoriginally described by Kohler and Milstein (1975) Nature 256:495-497)(see also, Brown et al. (1981) J. Immunol. 127:539-46; Brown et al.(1980) J. Biol. Chem. 255:4980-83; Yeh et al. (1976) Proc. Natl. Acad.Sci. USA 76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75),the more recent human B cell hybridoma technique (Kozbor et al. (1983)Immunol. Today 4:72), the EBV-hybridoma technique (Cole et al. (1985),Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96)or trioma techniques. The technology for producing monoclonal antibodyhybridomas is well known (see generally R. H. Kenneth, in MonoclonalAntibodies: A New Dimension In Biological Analyses, Plenum PublishingCorp., New York, N.Y. (1980); E. A. Lerner (1981) Yale J. Biol. Med.,54:387-402; M. L. Gefter et al. (1977) Somatic Cell Genet. 3:231-36).Briefly, an immortal cell line (typically a myeloma) is fused tolymphocytes (typically splenocytes) from a mammal immunized with a CXCR4or c-Kit immunogen as described above, and the culture supernatants ofthe resulting hybridoma cells are screened to identify a hybridomaproducing a monoclonal antibody that binds CXCR4 or c-Kit.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating ananti-CXCR4 or c-Kit monoclonal antibody (see, i.e., G. Galfre et al.(1977) Nature 266:55052; Gefter et al. Somatic Cell Genet., cited supra;Lerner, Yale J. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies,cited supra). Moreover, the ordinarily skilled worker will appreciatethat there are many variations of such methods which also would beuseful. Typically, the immortal cell line (i.e., a myeloma cell line) isderived from the same mammalian species as the lymphocytes. For example,murine hybridomas can be made by fusing lymphocytes from a mouseimmunized with an immunogenic preparation of the present invention withan immortalized mouse cell line. Preferred immortal cell lines are mousemyeloma cell lines that are sensitive to culture medium containinghypoxanthine, aminopterin and thymidine (“HAT medium”). Any of a numberof myeloma cell lines can be used as a fusion partner according tostandard techniques, i.e., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 orSp2/O-Ag14 myeloma lines. These myeloma lines are available from ATCC.Typically, HAT-sensitive mouse myeloma cells are fused to mousesplenocytes using polyethylene glycol (“PEG”). Hybridoma cells resultingfrom the fusion are then selected using HAT medium, which kills unfusedand unproductively fused myeloma cells (unfused splenocytes die afterseveral days because they are not transformed). Hybridoma cellsproducing a monoclonal antibody of the invention are detected byscreening the hybridoma culture supernatants for antibodies that bindCXCR4 or c-Kit, i.e., using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-CXCR4 or c-Kit antibody can be identified and isolatedby screening a recombinant combinatorial immunoglobulin library (i.e.,an antibody phage display library) with CXCR4 or c-Kit to therebyisolate immunoglobulin library members that bind CXCR4 or c-Kit,respectively. Kits for generating and screening phage display librariesare commercially available (i.e., the Pharmacia Recombinant PhageAntibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP™Phage Display Kit, Catalog No. 240612). Additionally, examples ofmethods and reagents particularly amenable for use in generating andscreening antibody display library can be found in, for example, Ladneret al. U.S. Pat. No. 5,223,409; Kang et al. PCT InternationalPublication No. WO 92/18619; Dower et al. PCT International PublicationNo. WO 91/17271; Winter et al. PCT International Publication WO92/20791; Markland et al. PCT International Publication No. WO 92/15679;Breitling et al. PCT International Publication WO 93/01288; McCaffertyet al. PCT International Publication No. WO 92/01047; Garrard et al. PCTInternational Publication No. WO 92/09690; Ladner et al. PCTInternational Publication No. WO 90/02809; Fuchs et al. (1991)Bio/Technology 9:1369-1372; Hay et al. (1992) Hum. Antibod. Hybridomas3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al.(1993) EMBO J. 12:725-734; Hawkins et al. (1992) J. Mol. Biol.226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al.(1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrard et al. (1991)Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nucleic Acids Res.19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad. Sci. USA88:7978-7982; and McCafferty et al. Nature (1990) 348:552-554.

Additionally, recombinant anti-CXCR4 or c-Kit antibodies, such aschimeric and humanized monoclonal antibodies, comprising both human andnon-human portions, which can be made using standard recombinant DNAtechniques, are within the scope of the invention. Such chimeric andhumanized monoclonal antibodies can be produced by recombinant DNAtechniques known in the art, for example using methods described inRobinson et al. International Application No. PCT/US86/02269; Akira, etal. European Patent Application 184,187; Taniguchi, M., European PatentApplication 171,496; Morrison et al. European Patent Application173,494; Neuberger et al. PCT International Publication No. WO 86/01533;Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al. European PatentApplication 125,023; Better et al. (1988) Science 240:1041-1043; Liu etal. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J.Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA84:214-218; Nishimura et al. (1987) Cancer Res. 47:999-1005; Wood et al.(1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al.(1986) BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones et al.(1986) Nature 321:552-525; Verhoeyen et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060.

An anti-CXCR4 or c-Kit antibody (i.e., monoclonal antibody) can be usedto isolate CXCR4 or c-Kit by standard techniques, such as affinitychromatography or immunoprecipitation. An anti-CXCR4 or c-Kit antibodycan facilitate the purification of natural CXCR4 or c-Kit from cells andof recombinantly produced CXCR4 or c-Kit expressed in host cells.Moreover, an anti-CXCR4 or c-Kit antibody can be used to detect CXCR4 orc-Kit protein (i.e., in a cellular lysate or cell supernatant) in orderto evaluate the abundance and pattern of expression of the CXCR4 orc-Kit protein. Anti-CXCR4 or c-Kit antibodies can be used diagnosticallyto monitor protein levels in tissue as part of a clinical testingprocedure, i.e., to, for example, determine the efficacy of a giventreatment, regimen. Detection can be facilitated by coupling (i.e.,physically linking) the antibody to a detectable substance. Examples ofdetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude 125I, 131I, 35S or 3H.

III. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to the use of vectors,preferably expression vectors, containing a nucleic acid encoding CXCR4,c-Kit, CXCR4 modulators, c-Kit modulators, or portions thereof. As usedherein, the term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid”, which refers to a circular double stranded DNAloop into which additional DNA segments can be ligated. Another type ofvector is a viral vector, wherein additional DNA segments can be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (i.e.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (i.e., non-episomal mammalian vectors)are integrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “expression vectors”. In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids.In the present specification, “plasmid” and “vector” can be usedinterchangeably as the plasmid is the most commonly used form of vector.However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (i.e., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (i.e., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (i.e., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those which direct constitutive expression of anucleotide sequence in many types of host cell and those which directexpression of the nucleotide sequence only in certain host cells (i.e.,tissue-specific regulatory sequences). In a preferred embodiment, a lungspecific promoter is used to direct expression of the nucleotidesequence in lung cells. It will be appreciated by those skilled in theart that the design of the expression vector can depend on such factorsas the choice of the host cell to be transformed, the level ofexpression of protein desired, etc. The expression vectors of theinvention can be introduced into host cells to thereby produce proteinsor peptides, including fusion proteins or peptides, encoded by nucleicacids as described herein (i.e., CXCR4 or c-Kit proteins, mutant formsof CXCR4 or c-Kit, fusion proteins, etc.).

The recombinant expression vectors of the invention can be designed forexpression of CXCR4 or c-Kit in prokaryotic or eukaryotic cells. Forexample, CXCR4 or c-Kit can be expressed in bacterial cells such as E.coli, insect cells (using baculovirus expression vectors) yeast cells ormammalian cells. Suitable host cells are discussed further in Goeddel,Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990). Alternatively, the recombinant expressionvector can be transcribed and translated in vitro, for example using T7promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67:3140), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein. In oneembodiment, the coding sequence of the CXCR4 or c-Kit is cloned into apGEX expression vector to create a vector encoding a fusion proteincomprising, from the N-terminus to the C-terminus, GST-thrombin cleavagesite-CXCR4 or c-Kit. The fusion protein can be purified by affinitychromatography using glutathione-agarose resin. Recombinant CXCR4 orc-Kit unfused to GST can be recovered by cleavage of the fusion proteinwith thrombin.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studieret al., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 60-89). Target gene expression from thepTrc vector relies on host RNA polymerase transcription from a hybridtrp-lac fusion promoter. Target gene expression from the pET 11d vectorrelies on transcription from a T7 gn10-lac fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a resident λprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al. (1992) Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the CXCR4 or c-Kit expression vector is a yeastexpression vector. Examples of vectors for expression in yeast S.cerivisae include pYepSec1 (Baldari, et al., (1987) EMBO J. 6:229-234),pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz etal., (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, SanDiego, Calif.).

Alternatively, CXCR4 or c-Kit can be expressed in insect cells usingbaculovirus expression vectors. Baculovirus vectors available forexpression of proteins in cultured insect cells (i.e., Sf9 cells)include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165)and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, B. (1987) Nature329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When usedin mammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (i.e., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the muscle specific casein kinase promoter, thealbumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev.1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv.Immunol. 43:235-275), in particular promoters of T cell receptors(Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins(Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell33:741-748), neuron-specific promoters (i.e., the neurofilamentpromoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science230:912-916), and mammary gland-specific promoters (i.e., milk wheypromoter; U.S. Pat. No. 4,873,316 and European Application PublicationNo. 264,166). Developmentally-regulated promoters are also encompassed,for example the murine hox promoters (Kessel and Gruss (1990) Science249:374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989)Genes Dev. 3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to CXCR4 or c-Kit mRNA. Regulatory sequencesoperatively linked to a nucleic acid cloned in the antisense orientationcan be chosen which direct the continuous expression of the antisenseRNA molecule in a variety of cell types, for instance viral promotersand/or enhancers, or regulatory sequences can be chosen which directconstitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes see Weintraub, H. etal., Antisense RNA as a molecular tool for genetic analysis,Reviews—Trends in Genetics, Vol. 1(1) 1986.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example,CXCR4 or c-Kit protein can be expressed in bacterial cells such as E.coli, insect cells, yeast or mammalian cells (such as muscle cells,Chinese hamster ovary cells (CHO) or COS cells). Other suitable hostcells are known to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (i.e., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (i.e., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as that encoding CXCR4 or c-Kit or can be introduced on aseparate vector. Cells stably transfected with the introduced nucleicacid can be identified by drug selection (i.e., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

A host cell used in the methods of the invention, such as a prokaryoticor eukaryotic host cell in culture, can be used to produce (i.e.,express) CXCR4 or c-Kit protein. Accordingly, the invention furtherprovides methods for producing CXCR4 or c-Kit protein using the hostcells of the invention. In one embodiment, the method comprisesculturing the host cell of invention (into which a recombinantexpression vector encoding CXCR4 or c-Kit has been introduced) in asuitable medium until CXCR4 or c-Kit is produced. In another embodiment,the method further comprises isolating CXCR4 or c-Kit from the medium orthe host cell.

The host cells of the invention can also be used to produce nonhumantransgenic animals. The nonhuman transgenic animals (i.e., mice, rats,monkeys, horses, dogs, turkeys, fish, cows, pigs, sheep, goats, frogs,or chickens) can be used, for example, in screening assays designed toidentify agents or compounds, i.e., drugs, pharmaceuticals, etc., whichcan be used to modulate SCLC proliferation and/or metastasis. Forexample, in one embodiment, a host cell of the invention is a fertilizedoocyte or an embryonic stem cell into which CXCR4 or c-Kit codingsequences have been introduced. Such host cells can then be used tocreate non-human transgenic animals in which exogenous CXCR4 or c-Kitsequences have been introduced into their genome or homologousrecombinant animals in which endogenous CXCR4 or c-Kit sequences havebeen altered. Such animals are useful for studying the function and/oractivity of CXCR4 or c-Kit and for identifying and/or evaluatingmodulators of CXCR4 or c-Kit activity. As used herein, a “transgenicanimal” is a nonhuman animal, preferably a mammal, more preferably arodent such as a rat or mouse, in which one or more of the cells of theanimal includes a transgene. Other examples of transgenic animalsinclude nonhuman primates, sheep, dogs, cows, goats, chickens,amphibians, etc. A transgene is exogenous DNA which is integrated intothe genome of a cell from which a transgenic animal develops and whichremains in the genome of the mature animal, thereby directing theexpression of an encoded gene product in one or more cell types ortissues of the transgenic animal. As used herein, a “homologousrecombinant animal” is a nonhuman animal, preferably a mammal, morepreferably a mouse, in which an endogenous CXCR4 or c-Kit gene has beenaltered by homologous recombination between the endogenous gene and anexogenous DNA molecule introduced into a cell of the animal, i.e., anembryonic cell of the animal, prior to development of the animal.

A transgenic animal of the invention can be created by introducing CXCR4or c-Kit encoding nucleic acid into the male pronuclei of a fertilizedoocyte, i.e., by microinjection, retroviral infection, and allowing theoocyte to develop in a pseudopregnant female foster animal. The humanCXCR4 or c-Kit cDNA sequence can be introduced as a transgene into thegenome of a nonhuman animal. Alternatively, a nonhuman homologue of thehuman CXCR4 gene (SEQ ID NO:1) or c-Kit gene (SEQ ID NO:3), such as amouse CXCR4 or c-Kit gene, can used as a transgene. Intronic sequencesand polyadenylation signals can also be included in the transgene toincrease the efficiency of expression of the transgene. Atissue-specific regulatory sequence(s) can be operably linked to theCXCR4 or c-Kit transgene to direct expression of CXCR4 or c-Kit proteinto particular cells. Methods for generating transgenic animals viaembryo manipulation and microinjection, particularly animals such asmice, have become conventional in the art and are described, forexample, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder etal., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B.,Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1986). Similar methods are used for productionof other transgenic animals. A transgenic founder animal can beidentified based upon the presence of the CXCR4 or c-Kit transgene inits genome and/or expression of CXCR4 or c-Kit mRNA in tissues or cellsof the animals. A transgenic founder animal can then be used to breedadditional animals carrying the transgene. Moreover, transgenic animalscarrying a transgene encoding CXCR4 or c-Kit can further be bred toother transgenic animals carrying other transgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of a CXCR4 or c-Kit gene into which adeletion, addition or substitution has been introduced to thereby alter,i.e., functionally disrupt, the CXCR4 or c-Kit gene. The CXCR4 or c-Kitgene can be a human gene (i.e., from a human genomic clone isolated froma human genomic library screened with the cDNA of SEQ ID NO:1 or 3), butmore preferably, is a nonhuman homologue of a human CXCR4 or c-Kit gene.For example, a mouse CXCR4 or c-Kit gene can be used to construct ahomologous recombination vector suitable for altering an endogenousCXCR4 or c-Kit gene in the mouse genome. In a preferred embodiment, thevector is designed such that, upon homologous recombination, theendogenous CXCR4 or c-Kit gene is functionally disrupted (i.e., nolonger encodes a functional protein; also referred to as a “knock out”vector). Alternatively, the vector can be designed such that, uponhomologous recombination, the endogenous CXCR4 or c-Kit gene is mutatedor otherwise altered but still encodes functional protein (i.e., theupstream regulatory region can be altered to thereby alter theexpression of the endogenous CXCR4 or c-Kit protein). In the homologousrecombination vector, the altered portion of the CXCR4 or c-Kit gene isflanked at its 5′ and 3′ ends by additional nucleic acid of the CXCR4 orc-Kit gene to allow for homologous recombination to occur between theexogenous CXCR4 or c-Kit gene carried by the vector and an endogenousCXCR4 or c-Kit gene in an embryonic stem cell. The additional flankingCXCR4 or c-Kit nucleic acid is of sufficient length for successfulhomologous recombination with the endogenous gene. Typically, severalkilobases of flanking DNA (both at the 5′ and 3′ ends) are included inthe vector (see i.e., Thomas, K. R. and Capecchi, M. R. (1987) Cell51:503 for a description of homologous recombination vectors). Thevector is introduced into an embryonic stem cell line (i.e., byelectroporation) and cells in which the introduced CXCR4 or c-Kit genehas homologously recombined with the endogenous CXCR4 or c-Kit gene areselected (see i.e., Li, E. et al. (1992) Cell 69:915). The selectedcells are then injected into a blastocyst of an animal (i.e., a mouse)to form aggregation chimeras (see i.e., Bradley, A. in Teratocarcinomasand Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed.(IRL, Oxford, 1987) pp. 113-152). A chimeric embryo can then beimplanted into a suitable pseudopregnant female foster animal and theembryo brought to term. Progeny harboring the homologously recombinedDNA in their germ cells can be used to breed animals in which all cellsof the animal contain the homologously recombined DNA by germlinetransmission of the transgene. Methods for constructing homologousrecombination vectors and homologous recombinant animals are describedfurther in Bradley, A. (1991) Current Opinion in Biotechnology 2:823-829and in PCT International Publication Nos.: WO 90/11354 by Le Mouellec etal.; WO 91101140 by Smithies et al.; WO 92/0968 by Zijlstra et al.; andWO 93/04169 by Berns et al.,

In one embodiment of the invention, transgenic animals are created usinga vector containing a lung specific promoter operatively linked to aCXCR4 or c-Kit nucleic acid molecule.

In another embodiment, transgenic nonhuman animals can be produced whichcontain selected systems which allow for regulated expression of thetransgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, i.e., Lakso et al. (1992) Proc. Natl. Acad.Sci. USA 89:6232-6236. Another example of a recombinase system is theFLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.(1991) Science 251:1351-1355. If a cre/loxP recombinase system is usedto regulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein are required.Such animals can be provided through the construction of “double”transgenic animals, ie., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

Clones of the nonhuman transgenic animals described herein can also beproduced according to the methods described in Wilmut, I. et al. (1997)Nature 385:810-813 and PCT International Publication Nos. WO 97/07668and WO 97/07669. In brief, a cell, i.e., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter Go phase. The quiescent cell can then be fused, i.e., throughthe use of electrical pulses, to an enucleated oocyte from an animal ofthe same species from which the quiescent cell is isolated. Thereconstructed oocyte is then cultured such that it develops to morula orblastocyst and then transferred to pseudopregnant female foster animal.The offspring borne of this female foster animal will be a clone of theanimal from which the cell, i.e., the somatic cell, is isolated.

IV. Pharmaceutical Compositions

The CXCR4 and/or c-Kit modulators (also referred to herein as “activecompounds”) of the invention can be incorporated into pharmaceuticalcompositions suitable for administration to a subject, i.e., a human.Such compositions typically comprise the nucleic acid molecule, protein,modulator, or antibody and a pharmaceutically acceptable carrier. Asused herein the language “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration. Theuse of such media and agents for pharmaceutically active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active compound, such media can be used in thecompositions of the invention. Supplementary active compounds can alsobe incorporated into the compositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, i.e., intravenous, intradermal,subcutaneous, oral (Le., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (i.e., a CXCR4 modulator and/or a c-Kit modulator) in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle which contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, i.e., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (i.e.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

V. Gene Therapy

In one embodiment of the invention, the nucleic acid molecules used inthe methods of the invention can be inserted into vectors and used asgene therapy vectors. Gene therapy vectors can be delivered to a subjectby, for example, intravenous injection, local administration (see U.S.Pat. No. 5,328,470) or by stereotactic injection (see i.e., Chen et al.(1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceuticalpreparation of the gene therapy vector can include the gene therapyvector in an acceptable diluent, or can comprise a slow release matrixin which the gene delivery vehicle is imbedded. Alternatively, where thecomplete gene delivery vector can be produced intact from recombinantcells, i.e. retroviral vectors, the pharmaceutical preparation caninclude one or more cells which produce the gene delivery system.

Viral vectors include, for example, recombinant retroviruses,adenovirus, adeno-associated virus, and herpes simplex virus-1.Retrovirus vectors and adeno-associated virus vectors are generallyunderstood to be the recombinant gene delivery system of choice for thetransfer of exogenous genes in vivo, particularly into humans.Adenovirus preferentially targets the liver when administeredsystemically (greater than 90+%; (Antinozzi et al. (1999) Annu. Rev.Nutr. 19:511-544) for reasons that may have to do with the expression ofviral receptors or the lack of vascular barriers in the liver.Alternatively they can be used for introducing exogenous genes ex vivointo liver cells in culture. These vectors provide efficient delivery ofgenes into liver cells, and the transferred nucleic acids are stablyintegrated into the chromosomal DNA of the host cell.

A major prerequisite for the use of viruses is to ensure the safety oftheir use, particularly with regard to the possibility of the spread ofwild-type virus in the cell population. The development of specializedcell lines (termed “packaging cells”) which produce onlyreplication-defective retroviruses has increased the utility ofretroviruses for gene therapy, and defective retroviruses are wellcharacterized for use in gene transfer for gene therapy purposes (for areview see Miller, A. D. (1990) Blood 76:271). Thus, recombinantretrovirus can be constructed in which part of the retroviral codingsequence (gag, pol env) is replaced by a gene of interest rendering theretrovirus replication defective. The replication defective retrovirusis then packaged into virions which can be used to infect a target cellthrough the use of a helper virus by standard techniques. Protocols forproducing recombinant retroviruses and for infecting cells in vitro orin vivo with such viruses can be found in Current Protocols in MolecularBiology, Ausubel, F. M. et al. (eds.) Greene Publishing Associates,(1989), Sections 9.10-9.14 and other standard laboratory manuals.Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM whichare well known to those skilled in the art. Examples of suitablepackaging virus lines for preparing both ecotropic and amphotropicretroviral systems include ψCrip, ψCre, ψ2 and ψAm.

Furthermore, it has been shown that it is possible to limit theinfection spectrum of retroviruses and consequently of retroviral-basedvectors, by modifying the viral packaging proteins on the surface of theviral particle (see, for example PCT publications WO93/25234 andWO94/06920). For instance, strategies for the modification of theinfection spectrum of retroviral vectors include: coupling antibodiesspecific for cell surface antigens to the viral env protein (Roux et al.(1989) Proc. Natl. Acad. Sci. USA 86:9079-9083; Julan et al. (1992) J.Gen. Virol. 73:3251-3255; and Goud et al. (1983) Virology 163:251-254);or coupling cell surface receptor ligands to the viral env proteins(Neda et al. (1991) J. Biol. Chem. 266:14143-14146). Coupling can be inthe form of the chemical cross-linking with a protein or other variety(i.e. lactose to convert the env protein to an asialoglycoprotein), aswell as by generating fusion proteins (i.e. single-chain antibody/envfusion proteins). Thus, in a specific embodiment of the invention, viralparticles containing a nucleic acid molecule containing a gene ofinterest operably linked to appropriate regulatory elements, aremodified for example according to the methods described above, such thatthey can specifically target subsets of liver cells. For example, theviral particle can be coated with antibodies to surface molecule thatare specific to certain types of liver cells. This method isparticularly useful when only specific subsets of liver cells aredesired to be transfected.

Another viral gene delivery system useful in the present inventionublizes adenovirus-derived vectors. The genome of an adenovirus can bemanipulated such that it encodes and expresses a gene product ofinterest but is inactivated in terms of its ability to replicate in anormal lytic viral life cycle. See for example Berkner et al. (1988)Biotechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; andRosenfeld et al. (1992) Cell 68:143-155. Suitable adenoviral vectorsderived from the adenovirus strain Ad type 5 dl324 or other strains ofadenovirus (i.e., Ad2, Ad3, Ad7 etc.) are well known to those skilled inthe art. Recombinant adenoviruses can be advantageous in certaincircumstances in that they are not capable of infecting nondividingcells. Furthermore, the virus particle is relatively stable and amenableto purification and concentration, and as above, can be modified so asto affect the spectrum of infectivity. Additionally, introducedadenoviral DNA (and foreign DNA contained therein) is not integratedinto the genome of a host cell but remains episomal, thereby avoidingpotential problems that can occur as a result of insertional mutagenesisin situations where introduced DNA becomes integrated into the hostgenome (i.e., retroviral DNA). Moreover, the carrying capacity of theadenoviral genome for foreign DNA is large (up to 8 kilobases) relativeto other gene delivery vectors (Berkner et al. cited supra; Haj-Ahmandand Graham (1986) J. Virol. 57:267). Most replication-defectiveadenoviral vectors currently in use and therefore favored by the presentinvention are deleted for all or parts of the viral E1 and E3 genes butretain as much as 80% of the adenoviral genetic material (see, i.e.,Jones et al. (1979) Cell 16:683; Berkner et al., supra; and Graham etal. in Methods in Molecular Biology, E. J. Murray, Ed. (Humana, Clifton,N.J., 1991) vol. 7. pp. 109-127). Expression of the gene of interestcomprised in the nucleic acid molecule can be under control of, forexample, the E1A promoter, the major late promoter (MLP) and associatedleader sequences, the E3 promoter, or exogenously added promotersequences.

Yet another viral vector system useful for delivery of a nucleic acidmolecule comprising a gene of interest is the adeno-associated virus(MV). Adeno-associated virus is a naturally occurring defective virusthat requires another virus, such as an adenovirus or a herpes virus, asa helper virus for efficient replication and a productive life cycle.(For a review see Muzyczka et al. Curr. Topics Microbiol. Immunol.(1992) 158:97-129). Adeno-associated viruses exhibit a high frequency ofstable integration (see for example Flotte et al. (1992) Am. J. Respir.Cell. Mol. Biol. 7:349-356; Samulski et al. (1989) J. Virol.63:3822-3828; and McLaughlin et a. (1989) J. Virol. 62:1963-1973).Vectors containing as few as 300 base pairs of AAV can be packaged andcan integrate. Space for exogenous DNA is limited to about 4.5 kb. An MVvector such as that described in Tratschin et al. (1985) Mol. Cell.Biol. 5:3251-3260 can be used to introduce DNA into T cells. A varietyof nucleic acids have been introduced into different cell types using MVvectors (see for example Hermonat et al. (1984) Proc. Natl. Acad. Sci.USA 81:6466-6470; Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081;Wondisford et al. (1988) Mol. Endocrinol. 2:32-39; Tratschin et al.(1984) J. Virol. 51:611-619; and Flotte et al. (1993) J. Biol. Chem.268:3781-3790). Other viral vector systems that may have application ingene therapy have been derived from herpes virus, vaccinia virus, andseveral RNA viruses.

Still another viral vector system useful for delivery of a nucleic acidmolecule comprising a gene of interest include the Herpes simplex virustype 1 (HSV-1) amplicon vectors for transfer of a gene into muscle(Wang, Y. et al. (2002) Hum. Gene. Ther. 13(2):261-273);

Other methods relating to the use of viral vectors in gene therapy canbe found in, i.e., Kay, M. A. (1997) Chest 111(6 Supp.):138S-142S;Ferry, N. and Heard, J. M. (1998) Hum. Gene Ther. 9:1975-81; Shiratory,Y. et al. (1999) Liver 19:265-74; Oka, K. et al. (2000) Curr. Opin.Lipidol. 11:179-86; Thule, P. M. and Liu, J. M. (2000) Gene Ther.7:1744-52; Yang, N. S. (1992) Crit. Rev. Biotechnol. 12:335-56; Alt, M.(1995) J. Hepatol. 23:746-58; Brody, S. L. and Crystal, R. G. (1994)Ann. N.Y. Acad. Sci. 716:90-101; Strayer, D. S. (1999) Expert Opin.Invetig. Drugs 8:2159-2172; Smith-Arica, J. R. and Bartlett, J. S.(2001) Curr. Cardiol. Rep. 3:43-49; and Lee, H. C. et al. (2000) Nature408:483-8.

VI. Screening Assays

The nucleic acid molecules, polypeptides, polypeptide homologues,modulators, and antibodies described herein can be used in methods oftreatment as well as drug screening assays. The invention provides amethod (also referred to herein as a screening assay”) for identifyingmodulators, i.e., candidate or test compounds or agents (i.e., peptides,peptidomimetics, small molecules or other drugs) which bind to CXCR4 orc-Kit proteins, have an inhibitory effect on, for example, CXCR4 orc-Kit expression or CXCR4 or c-Kit activity, or have an inhibitoryeffect on, for example, the expression or activity of a CXCR4 or c-Kittarget molecule. Such modulators of CXCR4 or c-Kit may be used in themethods of the invention to modulate cellular proliferation, movement,motility, adhesion, shape, morphological change, and/or metastasis ofSCLC cells. Such modulators may be further used to treat a subject withSCLC.

In one embodiment, the invention provides assays for screening candidateor test compounds which are target molecules of a CXCR4 or c-Kit proteinor polypeptide or biologically active portion thereof. In anotherembodiment, the invention provides assays for screening candidate ortest compounds which bind to or modulate the activity of a CXCR4 orc-Kit protein or polypeptide or biologically active portion thereof. Thetest compounds of the present invention can be obtained using any of thenumerous approaches in combinatorial library methods known in the art,including: biological libraries; spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary approach is limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds (Lam, K. S. (1997) Anticancer Drug Des.12:45).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example, in: DeWitt et al. (1993) Proc. Natl.Acad. Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (i.e., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage(Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladnersupra.).

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses a CXCR4 or c-Kit protein or biologically active portionthereof is contacted with a test compound and the ability of the testcompound to modulate CXCR4 or c-Kit activity is determined. Determiningthe ability of the test compound to modulate CXCR4 activity can beaccomplished by monitoring, for example, SDF-1α induced PI3-K activity(e.g., Akt and/or p70 S6 kinase phosphorylation) or SDF-1α mediatedcellular proliferation, adhesion, motility, or cell shape change in acell which expresses CXCR4. The cell, for example, can be an SCLC cell,e.g., a primary tumor cell, or a cell from an SCLC tumor cell line.

The ability of the test compound to modulate CXCR4 or c-Kit binding to atarget molecule can also be determined. Determining the ability of thetest compound to modulate CXCR4 or c-Kit binding to a target molecule(e.g., SDF-1α or SCF, respectively) can be accomplished, for example, bycoupling the CXCR4 or c-Kit target molecule with a radioisotope orenzymatic label such that binding of the CXCR4 or c-Kit target moleculeto CXCR4 or c-Kit, respectively, can be determined by detecting thelabeled CXCR4 or c-Kit target molecule in a complex. Alternatively,CXCR4 or c-Kit could be coupled with a radioisotope or enzymatic labelto monitor the ability of a test compound to modulate CXCR4 or c-Kitbinding to a CXCR4 or c-Kit target molecule in a complex. Determiningthe ability of the test compound to bind CXCR4 or c-Kit can beaccomplished, for example, by coupling the compound with a radioisotopeor enzymatic label such that binding of the compound to CXCR4 or c-Kitcan be determined by detecting the labeled CXCR4 or c-Kit compound in acomplex. For example, compounds (i.e., CXCR4 or c-Kit target molecules)can be labeled with 125I, 35S, 14C, or 3H, either directly orindirectly, and the radioisotope detected by direct counting ofradioemission or by scintillation counting. Alternatively, compounds canbe enzymatically labeled with, for example, horseradish peroxidase,alkaline phosphatase, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product.

It is also within the scope of this invention to determine the abilityof a compound or target molecule to interact with CXCR4 or c-Kit withoutthe labeling of any of the interactants. For example, a microphysiometercan be used to detect the interaction of a compound with CXCR4 or c-Kitwithout the labeling of either the compound or the CXCR4 or c-Kit.McConnell, H. M. et al. (1992) Science 257:1906-1912. As used herein, a“microphysiometer” (i.e., Cytosensor) is an analytical instrument thatmeasures the rate at which a cell acidifies its environment using alight-addressable potentiometric sensor (LAPS). Changes in thisacidification rate can be used as an indicator of the interactionbetween a compound and CXCR4 or c-Kit.

In another embodiment, an assay is a cell-based assay comprisingcontacting a cell expressing a CXCR4 or c-Kit target molecule with atest compound and determining the ability of the test compound tomodulate (Le. stimulate or inhibit) the activity of the CXCR4 or c-Kittarget molecule. Determining the ability of the test compound tomodulate the activity of a CXCR4 or c-Kit target molecule can beaccomplished, for example, by determining the ability of a CXCR4 orc-Kit protein to bind to or interact with the CXCR4 or c-Kit targetmolecule, or by determining the ability of a CXCR4 or c-Kit protein toinduce expression from a reporter construct.

Determining the ability of the CXCR4 or c-Kit protein, or a biologicallyactive fragment thereof, to bind to or interact with a CXCR4 or c-Kittarget molecule can be accomplished by one of the methods describedabove for determining direct binding. In a preferred embodiment,determining the ability of the CXCR4 or c-Kit protein to bind to orinteract with a CXCR4 or c-Kit target molecule can be accomplished bydetermining the activity of the target molecule. For example, theactivity of the target molecule can be determined by detecting inductionof a cellular response (i.e., induction of PI3-K activity by SDF-1α orSCF), detecting catalytic/enzymatic activity of the target molecule uponan appropriate substrate, detecting the induction of a reporter gene(comprising a target-responsive regulatory element operatively linked toa nucleic acid encoding a detectable marker, i.e., luciferase), ordetecting a target-regulated cellular response (i.e., proliferation).

In yet another embodiment, an assay of the present invention is acell-free assay in which a CXCR4 or c-Kit protein or biologically activeportion thereof is contacted with a test compound and the ability of thetest compound to bind to the CXCR4 or c-Kit protein or biologicallyactive portion thereof is determined. Preferred biologically activeportions of the CXCR4 or c-Kit proteins to be used in assays of thepresent invention include fragments which participate in interactionswith target molecules (e.g., SDF-1α or SCF). Binding of the testcompound to the CXCR4 or c-Kit protein can be determined either directlyor indirectly as described above. In a preferred embodiment, the assayincludes contacting the CXCR4 or c-Kit protein or biologically activeportion thereof with a known compound which binds CXCR4 or c-Kit to forman assay mixture, contacting the assay mixture with a test compound, anddetermining the ability of the test compound to interact with a CXCR4 orc-Kit protein, wherein determining the ability of the test compound tointeract with a CXCR4 or c-Kit protein comprises determining the abilityof the test compound to preferentially bind to CXCR4 or c-Kit orbiologically active portion thereof as compared to the known compound.

In another embodiment, the assay is a cell-free assay in which a CXCR4or c-Kit protein or biologically active portion thereof is contactedwith a test compound and the ability of the test compound to modulate(i.e., stimulate or inhibit) the activity of the CXCR4 or c-Kit proteinor biologically active portion thereof is determined. Determining theability of the test compound to modulate the activity of a CXCR4 orc-Kit protein can be accomplished, for example, by determining theability of the CXCR4 or c-Kit protein to bind to a CXCR4 or c-Kit targetmolecule by one of the methods described above for determining directbinding. Determining the ability of the CXCR4 or c-Kit protein to bindto a CXCR4 or c-Kit target molecule can also be accomplished using atechnology such as real-time Biomolecular Interaction Analysis (BIA).Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 andSzabo et al. (1995) Curr. Opin. Struct Biol. 5:699-705. As used herein,“BIA” is a technology for studying biospecific interactions in realtime, without labeling any of the interactants (i.e., BIAcore). Changesin the optical phenomenon of surface plasmon resonance (SPR) can be usedas an indication of real-time reactions between biological molecules.

In an alternative embodiment, determining the ability of the testcompound to modulate the activity of a CXCR4 or c-Kit protein can beaccomplished by determining the ability of the CXCR4 or c-Kit protein tofurther modulate the activity of a downstream effector of a CXCR4 orc-Kit target molecule. For example, the activity of the effectormolecule on an appropriate target can be determined or the binding ofthe effector to an appropriate target can be determined as previouslydescribed.

In yet another embodiment, the cell-free assay involves contacting aCXCR4 or c-Kit protein or biologically active portion thereof with aknown compound which binds the CXCR4 or c-Kit protein (i.e., SDF-1α orSCF, respectively) to form an assay mixture, contacting the assaymixture with a test compound, and determining the ability of the testcompound to interact with the CXCR4 protein, wherein determining theability of the test compound to interact with the CXCR4 or c-Kit proteincomprises determining the ability of the CXCR4 or c-Kit protein topreferentially bind to or modulate the activity of a CXCR4 or c-Kittarget molecule.

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize either CXCR4 or c-Kit orits target molecule to facilitate separation of complexed fromuncomplexed forms of one or both of the proteins, as well as toaccommodate automation of the assay. Binding of a test compound to aCXCR4 or c-Kit protein, or interaction of a CXCR4 or c-Kit protein witha target molecule in the presence and absence of a candidate compound,can be accomplished in any vessel suitable for containing the reactants.Examples of such vessels include microtiter plates, test tubes, andmicro-centrifuge tubes. In one embodiment, a fusion protein can beprovided which adds a domain that allows one or both of the proteins tobe bound to a matrix. For example, glutathione-5-transferase/CXCR4 orc-Kit fusion proteins or glutathione-5-transferase/target fusionproteins can be adsorbed onto glutathione sepharose beads (SigmaChemical, St. Louis, Mo.) or glutathione derivatized micrometer plates,which are then combined with the test compound or the test compound andeither the non-adsorbed target protein or CXCR4 or c-Kit protein, andthe mixture incubated under conditions conducive to complex formation(i.e., at physiological conditions for salt and pH). Followingincubation, the beads or microtiter plate wells are washed to remove anyunbound components, the matrix immobilized in the case of beads, complexdetermined either directly or indirectly, for example, as describedabove. Alternatively, the complexes can be dissociated from the matrix,and the level of CXCR4 or c-Kit binding or activity determined usingstandard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either a CXCR4 orc-Kit protein or a CXCR4 or c-Kit target molecule can be immobilizedutilizing conjugation of biotin and streptavidin. Biotinylated CXCR4 orc-Kit protein or target molecules can be prepared frombiotin-NHS(N-hydroxy-succinimide) using techniques known in the art(i.e., biotinylation kit, Pierce Chemicals, Rockford, Ill.), andimmobilized in the wells of streptavidin-coated 96 well plates (PierceChemical). Alternatively, antibodies reactive with CXCR4 or c-Kitprotein or target molecules but which do not interfere with binding ofthe CXCR4 or c-Kit protein to its target molecule can be derivatized tothe wells of the plate, and unbound target or CXCR4 or c-Kit proteintrapped in the wells by antibody conjugation. Methods for detecting suchcomplexes, in addition to those described above for the GST-immobilizedcomplexes, include immunodetection of complexes using antibodiesreactive with the CXCR4 or c-Kit protein or target molecule, as well asenzyme-linked assays which rely on detecting an enzymatic activityassociated with the CXCR4 or c-Kit protein or target molecule.

In another embodiment, modulators of CXCR4 or c-Kit expression areidentified in a method wherein a cell is contacted with a candidatecompound and the expression of CXCR4 or c-Kit mRNA or protein in thecell is determined. The level of expression of CXCR4 or c-Kit mRNA orprotein in the presence of the candidate compound is compared to thelevel of expression of CXCR4 or c-Kit mRNA or protein in the absence ofthe candidate compound. The candidate compound can then be identified asa modulator of CXCR4 or c-Kit expression based on this comparison. Forexample, when expression of CXCR4 or c-Kit mRNA or protein is greater(statistically significantly greater) in the presence of the candidatecompound than in its absence, the candidate compound is identified as astimulator of CXCR4 or c-Kit mRNA or protein expression. Alternatively,when expression of CXCR4 or c-Kit mRNA or protein is less (statisticallysignificantly less) in the presence of the candidate compound than inits absence, the candidate compound is identified as an inhibitor ofCXCR4 or c-Kit mRNA or protein expression. The level of CXCR4 or c-KitmRNA or protein expression in the cells can be determined by methodsdescribed herein for detecting CXCR4 or c-Kit mRNA or protein.

In yet another aspect of the invention, the CXCR4 or c-Kit proteins canbe used as “bait proteins” in a two-hybrid assay or three-hybrid assay(see, i.e., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300) to identify other proteins which bindto or interact with CXCR4 or c-Kit (“CXCR4 or c-Kit-binding proteins” or“CXCR4 or c-Kit-bp”) and are involved in CXCR4 or c-Kit activity. SuchCXCR4 or c-Kit-binding proteins are also likely to be involved in thepropagation of signals by the CXCR4 or c-Kit proteins or CXCR4 or c-Kittargets as, for example, downstream elements of a CXCR4 orc-Kit-mediated signaling pathway. Alternatively, such CXCR4 orc-Kit-binding proteins may be CXCR4 or c-Kit inhibitors.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a CXCR4 or c-Kitprotein is fused to a gene encoding the DNA binding domain of a knowntranscription factor (i.e., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming a CXCR4 orc-Kit dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (i.e., LacZ) which is operablylinked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with the CXCR4 or c-Kit protein.

In another aspect, the invention pertains to a combination of two ormore of the assays described herein. For example, a modulating agent canbe identified using a cell-based or a cell-free assay, and the abilityof the agent to modulate the activity of a CXCR4 or c-Kit protein can beconfirmed in vivo, i.e., in an animal such as a mouse model for SCLC.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (i.e., a CXCR4 or c-Kit modulating agent, an antisenseCXCR4 or c-Kit nucleic acid molecule, a CXCR4 or c-Kit specificantibody, or a CXCR4 or c-Kit binding partner) can be used in an animalmodel to determine the efficacy, toxicity, or side effects of treatmentwith such an agent. Alternatively, an agent identified as describedherein can be used in an animal model to determine the mechanism ofaction of such an agent. Furthermore, this invention pertains to uses ofnovel agents identified by the above-described screening assays fortreatments as described herein.

In yet another embodiment, the invention provides a method foridentifying a compound (i.e., a screening assay) capable of use in thetreatment of SCLC. This method typically includes the step of assayingthe ability of the compound or agent to modulate the expression of theCXCR4 or c-Kit nucleic acid or the activity of the CXCR4 or c-Kitprotein thereby identifying a compound for treating SCLC. Methods forassaying the ability of the compound or agent to modulate the expressionof the CXCR4 or c-Kit nucleic acid or activity of the CXCR4 or c-Kitprotein are typically cell-based assays. For example, cells which aresensitive to ligands which transduce signals via a pathway involvingCXCR4 or c-Kit can be induced to overexpress a CXCR4 or c-Kit protein inthe presence and absence of a candidate compound. Candidate compoundswhich produce a statistically significant change in CXCR4 or c-Kitdependent responses (either stimulation or inhibition) can beidentified. In one embodiment, expression of the CXCR4 or c-Kit nucleicacid or activity of a CXCR4 or c-Kit protein is modulated in cells andthe effects of candidate compounds on the readout of interest (such asrate of cell proliferation or differentiation) are measured. Forexample, the expression or activity of genes which are up- ordown-regulated in response to a CXCR4 protein-dependent signal cascadecan be assayed (e.g., the phosphorylation of Akt and/or p70 S6 kinase).In preferred embodiments, the regulatory regions of such genes, i.e.,the 5′ flanking promoter and enhancer regions, are operably linked to adetectable marker (such as luciferase) which encodes a gene product thatcan be readily detected. Phosphorylation of CXCR4 or c-Kit or CXCR4 orc-Kit target molecules can also be measured, for example, byimmunoblotting.

Alternatively, modulators of CXCR4 or c-Kit nucleic acid expression(i.e., compounds which can be used to treat SCLC) can be identified in amethod wherein a cell is contacted with a candidate compound and theexpression of CXCR4 or c-Kit mRNA or protein in the cell is determined.The level of expression of CXCR4 or c-Kit mRNA or protein in thepresence of the candidate compound is compared to the level ofexpression of CXCR4 or c-Kit mRNA or protein in the absence of thecandidate compound. The candidate compound can then be identified as amodulator of CXCR4 or c-Kit nucleic acid expression based on thiscomparison and be used to treat a disorder characterized by aberrantCXCR4 or c-Kit nucleic acid expression. For example, when expression ofCXCR4 or c-Kit mRNA or polypeptide is greater (statisticallysignificantly greater) in the presence of the candidate compound than inits absence, the candidate compound is identified as a stimulator ofCXCR4 or c-Kit nucleic acid expression. Alternatively, when CXCR4 orc-Kit nucleic acid expression is less (statistically significantly less)in the presence of the candidate compound than in its absence, thecandidate compound is identified as an inhibitor of CXCR4 or c-Kitnucleic acid expression. The level of CXCR4 or c-Kit nucleic acidexpression in the cells can be determined by methods described hereinfor detecting CXCR4 or c-Kit mRNA or protein.

Modulators of CXCR4 or c-Kit protein activity and/or CXCR4 or c-Kitnucleic acid expression identified according to these drug screeningassays can be used to treat SCLC. Modulators of CXCR4 or c-Kit proteinactivity and/or CXCR4 or c-Kit nucleic acid expression may also be usedto treat disorders related to other functions of CXCR4 or c-Kitunrelated to SCLC (e.g., AIDS). These methods of treatment include thesteps of administering the modulators of CXCR4 or c-Kit protein activityand/or nucleic acid expression, i.e., in a pharmaceutical composition asdescribed in subsection IV above, to a subject in need of suchtreatment, ie., a subject with a disorder described herein.

VII. Monitoring the Effectiveness of an Anti-SCLC Agent

The presence or activity level of CXCR4 and/or c-Kit may be used to: 1)determine if SCLC can be or is likely to be successfully treated by anagent or combination of agents; 2) determine if SCLC is responding totreatment with an agent or combination of agents; 3) select anappropriate agent or combination of agents for treating SCLC; 4) monitorthe effectiveness of an ongoing treatment; and/or 5) identify newtreatments (either single agent or combination of agents). Inparticular, CXCR4 and/or c-Kit may be utilized as markers (surrogateand/or direct) to determine appropriate therapy, to monitor clinicaltherapy and human trials of a drug being tested for efficacy, and todevelop new agents and therapeutic combinations.

Accordingly, the present invention provides methods for determiningwhether an agent, e.g., a chemotherapeutic agent, can be used to inhibitproliferation or metastasis of SCLC comprising the steps of:

-   -   a) obtaining a sample of lung cells;    -   b) determining whether the cells express CXCR4;        thereby determining that the agent can be used to inhibit        proliferation or metastasis of small cell lung cancer when CXCR4        is expressed. In a further embodiment, the method comprises        determining whether the cells express c-Kit, thereby determining        that the agent can be used to inhibit proliferation or        metastasis of small cell lung cancer when CXCR4 and c-Kit are        expressed.        In another embodiment, the invention provides a method for        determining whether a patient would benefit from treatment with        an agent that inhibits CXCR4 comprising:    -   a) obtaining a lung sample from the patient; and    -   b) determining whether CXCR4 is expressed in the sample. In        another embodiment, the invention provides a method for        determining whether a patient would benefit from treatment with        an agent that inhibits CXCR4 and an agent that inhibits c-Kit        comprising:    -   a) obtaining a lung sample from the patient;    -   b) determining whether CXCR4 is expressed in the sample; and    -   c) determining whether c-Kit is expressed in the sample.        The activity level of CXCR4 and/or c-Kit can also be used to        assess whether SCLC has become refractory to an ongoing        treatment (e.g., a chemotherapeutic treatment). When SCLC is no        longer responding to a treatment the activity profile of the        cells will change: the level of activity of CXCR4 and/or c-Kit        will be reduced. Accordingly, in another embodiment, the        invention provides a method for determining whether treatment        with a CXCR4 inhibitor should be continued or discontinued in an        SCLC patient, comprising:    -   a) obtaining two or more samples comprising cells from a patient        during the course of treatment;    -   b) determining the level of activity in the cells of CXCR4; and    -   c) continuing treatment when the activity of CXCR4 does not        increase during treatment In another embodiment, the invention        provides a method for determining whether treatment with a        combination of a CXCR4 inhibitor and a receptor tyrosine kinase        inhibitor should be continued or discontinued in a small cell        lung cancer patient, comprising:    -   a) obtaining two or more samples comprising cells from a patient        during the course of treatment;    -   b) determining the level of activity in the cells of CXCR4;    -   c) determining the level of activity in the cells of c-Kit; and    -   d) continuing treatment when the activity levels of CXCR4 and/or        c-Kit do not increase during treatment.

This embodiment of the present invention relies on comparing two or moresamples obtained from a patient undergoing anti-SCLC treatment. It willbe appreciated that the samples may be from the lung of the patent (e.g.SCLC cells, tumor cells, etc.), obtained using standard methods. Ingeneral, it is preferable to obtain a first sample from the patientprior to beginning therapy and one or more samples during treatment. Insuch a use, a baseline of activity prior to therapy is determined andthen changes in the baseline state of expression is monitored during thecourse of therapy. Alternatively, two or more successive samplesobtained during treatment can be used without the need of apre-treatment baseline sample. In such a use, the first sample obtainedfrom the subject is used as a baseline for determining whether theactivity of CXCR4 and/or c-Kit is increasing or decreasing.

In general, when monitoring the effectiveness of a therapeutictreatment, two or more samples from the patient are examined.Preferably, three or more successively obtained samples are used,including at least one pretreatment sample.

In one embodiment of the invention, the expression of CXCR4 and/or c-Kitis detected by measuring CXCR4 and/or c-Kit mRNA levels, respectively.In yet another embodiment of the invention, the expression of CXCR4and/or c-Kit is detected by measuring CXCR4 and/or c-Kit protein levels,respectively.

As used herein, the term “agent” is defined broadly as anything thatSCLC cells, may be exposed to in a therapeutic protocol, preferably anagent that has been identified as being effective for the treatment ofSCLC, e.g., using the methods described herein. In the context of thepresent invention, such agents include, but are not limited to,chemotherapeutic agents, radiation and ultraviolet light. In a preferredembodiment, the agent is a CXCR4 inhibitor (e.g., AMD-3100, ALX40-4C,T22, T140, Met-SDF-1beta, T134, AMD-3465, or an agent identified by thescreening methods described herein). In another embodiment, the agent isa receptor tyrosine kinase inhibitor, e.g., a c-Kit inhibitor. In apreferred embodiment, the c-Kit inhibitor is imatinib mesylate (STI571or Gleevec™). In a further preferred embodiment, the agent is acombination of a CXCR4 inhibitor and a receptor tyrosine kinaseinhibitor.

Further to the above, the language “chemotherapeutic agent” is intendedto include chemical reagents which inhibit the growth of proliferatingcells or tissues wherein the growth of such cells or tissues isundesirable. Chemotherapeutic agents are well known in the art (seee.g., Gilman A. G., et al., The Pharmacological Basis of Therapeutics,8th Ed., Sec 12:1202-1263 (1990)), and are typically used to treatneoplastic diseases. In a preferred embodiment, the chemotherapeuticagents used in the methods of the invention are chemotherapeutic agentsused to treat SCLC.

The methods of the present invention may also be used to modulate theproliferation, growth, movement, motility, adhesion, morphology, and/ormetastasis of any type of cancer or tumor cells that express CXCR4and/or c-Kit, e.g., SCLC cells. As used herein, cancer cells, includingtumor cells, refer to cells that divide at an abnormal (increased) rate.

The source of the lung or SCLC cells used in the present method will bebased on how the method of the present invention is being used. Forexample, if the method is being used to determine whether a patient'sSCLC can be treated with an agent, or a combination of agents, then thepreferred source of cells will be lung cancer cells obtained from lungbiopsy from the patient, e.g., a tumor biopsy. If the method is beingused to monitor the effectiveness of a therapeutic protocol, then a lungtissue sample from the patient being treated is the preferred source. Ifthe method is being used to identify new therapeutic agents orcombinations, any SCLC cells, e.g., primary SCLC cells or an SCLC cellline, can be used.

A skilled artisan can readily select and obtain the appropriate cellsthat are used in the present method. For cancer cell lines, sources suchas The National Cancer Institute, for the NCI-H69 cells, are preferred.For cancer cells obtained from a patient, standard biopsy methods can beemployed.

In the methods of the present invention, the expression or level ofactivity of CXCR4 and/or c-Kit is determined. As used herein, theexpression of CXCR4 or c-Kit refers to whether or not CXCR4 or c-KitmRNA is expressed in the cells, e.g., the SCLC cells. As used herein,the level of activity of CXCR4 refers to, for example, the ability ofSDF-1α to modulate proliferation, adhesion, motility, cell shape, orPI3-K activity in the cells. As used herein, the level of activity ofc-Kit refers to, for example, the ability of SCF to modulateproliferation, adhesion, motility, cell shape, or PI3-K activity in thecells.

As used herein, a patient refers to any subject undergoing treatment forlung cancer (e.g., SCLC). The preferred subject will be a human patientundergoing chemotherapy treatment.

An exemplary method for detecting the presence or absence of apolypeptide or nucleic acid corresponding to CXCR4 and/or c-Kit in abiological sample involves obtaining a biological sample (e.g., a lungsample or a lung tumor sample) from a test subject and contacting thebiological sample with a compound or an agent capable of detecting thepolypeptide or nucleic acid (e.g., mRNA, genomic DNA, or cDNA). Thedetection methods of the invention can thus be used to detect mRNA,protein, cDNA, or genomic DNA, for example, in a biological sample invitro as well as in vivo. For example, in vitro techniques for detectionof mRNA include Northern hybridizations and in situ hybridizations. Invitro techniques for detection of a polypeptide corresponding to CXCR4or c-Kit include enzyme linked immunosorbent assays (ELISAs), Westernblots, immunoprecipitations and immunofluorescence. In vivo techniquesfor detection of genomic DNA include Southern hybridizations.Furthermore, in vivo techniques for detection of a polypeptidecorresponding to CXCR4 or c-Kit include introducing into a subject alabeled antibody directed against the polypeptide. For example, theantibody can be labeled with a radioactive marker whose presence andlocation in a subject can be detected by standard imaging techniques.

A general principle of such diagnostic and prognostic assays involvespreparing a sample or reaction mixture that may contain CXCR4 and/orc-Kit, and a probe, under appropriate conditions and for a timesufficient to allow CXCR4 and/or c-Kit and the probe to interact andbind, thus forming a complex that can be removed and/or detected in thereaction mixture. These assays can be conducted in a variety of ways.

For example, one method to conduct such an assay would involve anchoringCXCR4 and/or c-Kit or the probe onto a solid phase support, alsoreferred to as a substrate, and detecting target CXCR4 and/or c-Kitprobe complexes anchored on the solid phase at the end of the reaction.In one embodiment of such a method, a sample from a subject, which is tobe assayed for presence of CXCR4 and/or c-Kit, can be anchored onto acarrier or solid phase support. In another embodiment, the reversesituation is possible, in which the probe can be anchored to a solidphase and a sample from a subject can be allowed to react as anunanchored component of the assay.

There are many established methods for anchoring assay components to asolid phase. These include, without limitation, CXCR4 and/or c-Kit orprobe molecules which are immobilized through conjugation of biotin andstreptavidin. Such biotinylated assay components can be prepared frombiotin-NHS(N-hydroxy-succinimide) using techniques known in the art(e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), andimmobilized in the wells of streptavidin-coated 96 well plates (PierceChemical). In certain embodiments, the surfaces with immobilized assaycomponents can be prepared in advance and stored.

Other suitable carriers or solid phase supports for such assays includeany material capable of binding the class of molecule to which the CXCR4and/or c-Kit or probe belongs. Well-known supports or carriers include,but are not limited to, glass, polystyrene, nylon, polypropylene, nylon,polyethylene, dextran, amylases, natural and modified celluloses,polyacrylamides, gabbros, and magnetite.

In order to conduct assays with the above mentioned approaches, thenon-immobilized component is added to the solid phase upon which thesecond component is anchored. After the reaction is complete,uncomplexed components may be removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized uponthe solid phase. The detection of CXCR4 and/or c-Kit/probe complexesanchored to the solid phase can be accomplished in a number of methodsoutlined herein.

In a preferred embodiment, the probe, when it is the unanchored assaycomponent, can be labeled for the purpose of detection and readout ofthe assay, either directly or indirectly, with detectable labelsdiscussed herein and which are well-known to one skilled in the art.

It is also possible to directly detect CXCR4 and/or c-Kit/probe complexformation without further manipulation or labeling of either component(marker or probe), for example by utilizing the technique offluorescence energy transfer (see, for example, Lakowicz et al., U.S.Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No. 4,868,103). Afluorophore label on the first, ‘donor’ molecule is selected such that,upon excitation with incident light of appropriate wavelength, itsemitted fluorescent energy will be absorbed by a fluorescent label on asecond ‘acceptor’ molecule, which in turn is able to fluoresce due tothe absorbed energy. Alternately, the ‘donor’ protein molecule maysimply utilize the natural fluorescent energy of tryptophan residues.Labels are chosen that emit different wavelengths of light, such thatthe ‘acceptor’ molecule label may be differentiated from that of the‘donor’. Since the efficiency of energy transfer between the labels isrelated to the distance separating the molecules, spatial relationshipsbetween the molecules can be assessed. In a situation in which bindingoccurs between the molecules, the fluorescent emission of the ‘acceptor’molecule label in the assay should be maximal. An FET binding event canbe conveniently measured through standard fluorometric detection meanswell known in the art (e.g., using a fluorimeter).

In another embodiment, determination of the ability of a probe torecognize CXCR4 and/or c-Kit can be accomplished without labeling eitherassay component (probe or marker) by utilizing a technology such asreal-time Biomolecular Interaction Analysis (BIA).

Alternatively, in another embodiment, analogous diagnostic andprognostic assays can be conducted with CXCR4 and/or c-Kit and probe assolutes in a liquid phase. In such an assay, the complexed CXCR4 and/orc-Kit and probe are separated from uncomplexed components by any of anumber of standard techniques, including but not limited to:differential centrifugation, chromatography, electrophoresis andimmunoprecipitation. In differential centrifugation, marker/probecomplexes may be separated from uncomplexed assay components through aseries of centrifugal steps, due to the different sedimentationequilibria of complexes based on their different sizes and densities(see, for example, Rivas, G. and Minton, A. P. (1993) Trends BiochemSci. 18(8):284-7). Standard chromatographic techniques may also beutilized to separate complexed molecules from uncomplexed ones. Forexample, gel filtration chromatography separates molecules based onsize, and through the utilization of an appropriate gel filtration resinin a column format, for example, the relatively larger complex may beseparated from the relatively smaller uncomplexed components. Similarly,the relatively different charge properties of the CXCR4 and/orc-Kit/probe complex as compared to the uncomplexed components may beexploited to differentiate the complex from uncomplexed components, forexample through the utilization of ion-exchange chromatography resins.Such resins and chromatographic techniques are well known to one skilledin the art (see, e.g., Heegaard, N. H., 1998, J. Mol. Recognit. Winter11(1-6):141-8; Hage, D. S., and Tweed, S. A. J Chromatogr B Biomed SciAppl 1997 Oct. 10; 699(1-2):499-525). Gel electrophoresis may also beemployed to separate complexed assay components from unbound components(see, e.g., Ausubel et al., ed., Current Protocols in Molecular Biology,John Wiley & Sons, New York, 1987-1999). In this technique, protein ornucleic acid complexes are separated based on size or charge, forexample. In order to maintain the binding interaction during theelectrophoretic process, non-denaturing gel matrix materials andconditions in the absence of reducing agent are typically preferred.Appropriate conditions to the particular assay and components thereofwill be well known to one skilled in the art.

In a particular embodiment, the level of mRNA corresponding to CXCR4and/or c-Kit can be determined both by in situ and by in vitro formatsin a biological sample using methods known in the art. The term“biological sample” is intended to include tissues, cells, biologicalfluids and isolates thereof, isolated from a subject, as well astissues, cells and fluids present within a subject. In a preferredembodiment, a biological sample is preferably a lung sample or a lungtumor sample. Many expression detection methods use isolated RNA For invitro methods, any RNA isolation technique that does not select againstthe isolation of mRNA can be utilized for the purification of RNA fromovarian cells (see, e.g., Ausubel et al., ed., Current Protocols inMolecular Biology, John Wiley & Sons, New York 1987-1999). Additionally,large numbers of tissue samples can readily be processed usingtechniques well known to those of skill in the art, such as, forexample, the single-step RNA isolation process of Chomczynski (1989,U.S. Pat. No. 4,843,155).

The isolated mRNA can be used in hybridization or amplification assaysthat include, but are not limited to, Southern or Northern analyses,polymerase chain reaction analyses and probe arrays. One preferreddiagnostic method for the detection of mRNA levels involves contactingthe isolated mRNA with a nucleic acid molecule (probe) that canhybridize to the mRNA encoded by the gene being detected. The nucleicacid probe can be, for example, a full-length cDNA, or a portionthereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250or 500 nucleotides in length and sufficient to specifically hybridizeunder stringent conditions to a mRNA or genomic DNA encoding a marker ofthe present invention. Other suitable probes for use in the diagnosticassays of the invention are described herein. Hybridization of an mRNAwith the probe indicates that the marker in question (e.g., CXCR4 and/orc-Kit) is being expressed.

In one format, the mRNA is immobilized on a solid surface and contactedwith a probe, for example by running the isolated mRNA on an agarose geland transferring the mRNA from the gel to a membrane, such asnitrocellulose. In an alternative format, the probe(s) are immobilizedon a solid surface and the mRNA is contacted with the probe(s), forexample, in an Affymetrix gene chip array. A skilled artisan can readilyadapt known mRNA detection methods for use in detecting the level ofCXCR4 and/or c-Kit mRNA.

An alternative method for determining the level of mRNA corresponding toCXCR4 and/or c-Kit in a sample involves the process of nucleic acidamplification, e.g., by rtPCR (the experimental embodiment set forth inMullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany,1991, Proc. Natl. Acad. Sci. USA, 88:189-193), self sustained sequencereplication (Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh et al., 1989,Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal., 1988, Bio/Technology 6:1197), rolling circle replication (Lizardiet al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplificationmethod, followed by the detection of the amplified molecules usingtechniques well known to those of skill in the art. These detectionschemes are especially useful for the detection of nucleic acidmolecules if such molecules are present in very low numbers. As usedherein, amplification primers are defined as being a pair of nucleicacid molecules that can anneal to 5′ or 3′ regions of a gene (plus andminus strands, respectively, or vice-versa) and contain a short regionin between. In general, amplification primers are from about 10 to 30nucleotides in length and flank a region from about 50 to 200nucleotides in length. Under appropriate conditions and with appropriatereagents, such primers permit the amplification of a nucleic acidmolecule comprising the nucleotide sequence flanked by the primers.

For in situ methods, mRNA does not need to be isolated from the lungcells prior to detection. In such methods, a lung tumor or tissue sampleis prepared/processed using known histological methods. The sample isthen immobilized on a support, typically a glass slide, and thencontacted with a probe that can hybridize to mRNA that encodes CXCR4and/or c-Kit.

In another embodiment of the present invention, a polypeptidecorresponding to CXCR4 and/or c-Kit is detected. A preferred agent fordetecting a polypeptide of the invention is an antibody or antibodyfragment capable of binding to CXCR4 and/or c-Kit, preferably anantibody with a detectable label. Antibodies can be polyclonal, or morepreferably, monoclonal. An intact antibody, or a fragment thereof (e.g.,Fab or F(ab′)₂) can be used. The term “labeled”, with regard to theprobe or antibody, is intended to encompass direct labeling of the probeor antibody by coupling (i.e., physically linking) a detectablesubstance to the probe or antibody, as well as indirect labeling of theprobe or antibody by reactivity with another reagent that is directlylabeled. Examples of indirect labeling include detection of a primaryantibody using a fluorescently labeled secondary antibody andend-labeling of a DNA probe with biotin such that it can be detectedwith fluorescently labeled streptavidin.

A variety of formats can be employed to determine whether a samplecontains a protein (e.g., CXCR4 and/or c-Kit) that binds to a givenantibody. Examples of such formats include, but are not limited to,enzyme immunoassay (EIA), radioimmunoassay (RIA), Western blot analysisand enzyme linked immunoabsorbant assay (ELISA). A skilled artisan canreadily adapt known protein/antibody detection methods for use indetermining whether lung cells express CXCR4 and/or c-Kit.

In one format, antibodies, or antibody fragments, can be used in methodssuch as Western blots or immunofluorescence techniques to detect theexpressed proteins. In such uses, it is generally preferable toimmobilize either the antibody or proteins on a solid support. Suitablesolid phase supports or carriers include any support capable of bindingan antigen or an antibody. Well-known supports or carriers includeglass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite.

One skilled in the art will know many other suitable carriers forbinding antibody or antigen, and will be able to adapt such support foruse with the present invention. For example, protein isolated from lungcells can be run on a polyacrylamide gel electrophoresis and immobilizedonto a solid phase support such as nitrocellulose. The support can thenbe washed with suitable buffers followed by treatment with thedetectably labeled antibody. The solid phase support can then be washedwith the buffer a second time to remove unbound antibody. The amount ofbound label on the solid support can then be detected by conventionalmeans.

This invention is further illustrated by the following examples whichshould not be construed as limiting.

EXAMPLES

Materials and Methods

Cell Lines and Cell Culture

Ten small cell lung cancer (SCLC) cell lines (NCI-H69, NCI-H82,NCI-H128, NCI-H146, NCI-H209, NCI-H249, NCI-H345, NCI-H446, NCI-H510,and NCI-H526) were purchased from the American Type Culture Collection(Rockville, Md.) and maintained in RPMI 1640 medium (Cellgro)supplemented with 10% (v/v) fetal calf serum (FCS). MO7e cells weremaintained as described in Sattler, M. et al. (1997) J. Biol. Chem.272:10248-53. All cell lines were incubated at 37° C. in a 5% CO₂humidified atmosphere and harvested during log phase growth. Cells weredeprived of growth factors by incubation in RPMI 1640 medium containing0.5% (w/v) bovine serum albumin (BSA) (Sigma, St. Louis, Mo.) for 18hours. In some experiments, cells were treated with 5 μM STI571(Gleevec™, Novartis Pharmaceuticals, Basel, Switzerland) or 25 μMLY294002 (Sigma). Recombinant human SDF-1α and SCF (BioSourceInternational, Inc., Camarillo, Calif.) were used in the conditionsindicated in each experiment.

Cell Viability Assay

NCI-H69 cells (1×10⁶/ml) were cultured in serum-free (0.5% BSA) orserum-containing (0.5, 1, 5, or 10% FCS) media with or without 100 ng/mlSCF and/or SDF-1α. Viable cells were counted by trypan blue dyeexclusion test every 24 hours up to 72 hours. Data was plotted as themean±SD from three independent experiments. Student's t test was usedfor the statistical analysis of viable cell number under individualconditions. Differences were considered statistically significant atp<0.05.

RNase Protection Assay (RPA)

Chemokine receptor mRNA was detected using the hCR-6 multi-probetemplate set (RiboQuant, PharMingen, San Diego, Calif.) according to themanufacturer's protocol. This set contains DNA templates for CXCR-1, -2,-3, -4, Burkitt's lymphoma receptor (BLR)-1/CXCR5, BLR-2/CCR7,V28/CX3CR1, as well as ribosomal protein L32 and GAPDH(glyceradehyde-3-phosphate dehydrogenase) as controls. In brief,anti-sense RNA probes were generated from DNA templates by T7 RNApolymerase in the presence of [α-³²P] UTP (3,000 Ci/mmol; Life ScienceProducts, Inc., Boston, Mass.). Labeled probes were hybridized overnightat 56° C. with 20 μg total RNA isolated using RNeasy kit (Qiagen,Hilden, Germany). As the negative and positive controls, 2 μg yeast tRNAand human control RNA-2 were used, respectively. Unhybridized RNA wasdigested with RNase A and T1. RNase-protected probes were resolved on adenaturing 5% acrylamide-urea sequencing gel and identified byautoradiography.

FACS Analysis

Cells (1×10⁵) were washed three times in phosphate buffered saline (PBS)containing 0.5% BSA (PBS buffer), then incubated for 30 minutes at 4° C.with 10 μg/ml phycoerythrin (PE)-conjugated mouse-anti-human CXCR4monoclonal antibody or PE-labeled mouse control IgG₂₈ (R&D Systems Inc.,Minneapolis, Minn.). After washing the cells twice with PBS buffer toremove unbound antibodies, the stained cells were resuspended in 300 μlof PBS and analyzed by FACScan using Cell Quest software (BectonDickinson Labware, Franklin Lakes, N.J.).

Adhesion Assay

The wells of a 96-well tissue culture plate (Corning-Costar, Cambridge,Mass.) pre-coated with 10 μg/ml human plasma fibronectin (FN) or humancollagen type IV (col. IV) (Gibco BRL, Rockville, Md.) overnight at 4°C. were washed with PBS twice and blocked for 1 hour at 37° C. with RPMI1640 medium containing 0.2% BSA (adhesion media) before plating cells.NCI-H446 cells (3×10⁵) were washed twice, resuspended in the adhesionmedia with or without SDF-1α (100 ng/ml), and plated onto uncoated, FN-,or col. IV-coated wells. Unattached cells were removed after incubationfor 2 hours at 37° C. by gentle washing with adhesion media. Therelative number of attached viable cells was determined by the MTTcolorimetric assay (Sigma) following the instruction manual. Student's ttest was used for the statistical analysis of the attached cell numberand differences were taken significant at p<0.05.

Phase Contrast Microscopy for Cell Morphology

Serum-starved NCI-H69 cells (1×10⁶) were placed onto 6 well tissueculture plates (Becton Dickinson Labware) and incubated in serum freemedia in the absence or presence of STI571 (5 μM), SDF-1α (100 ng/ml)and/or SCF (100 ng/ml). Phase-contrast photomicrographs were taken atvarious time points (0, 1, 2, 4, 8, and 24 hours). For LY294002 studies,starved NCI-H446 cells (1×10⁶) were resuspended in serum free media andplaced onto a 35 mm tissue culture dish (Becton Dickinson Labware).After 24 hours incubation in the absence or presence of LY294002 (25 μM)and/or SDF-1α (100 ng/ml), phase-contrast pictures were taken.

Analysis of Cell Motility by Time-Lapse Video Microscopy (TLVM)

NCI-H446 cells were plated as above and the dish was placed into atemperature controlled chamber at 37° C. in an atmosphere of 5% CO₂. Thecells were examined by TLVM using an Olympus IX70 inverted microscope,Omega temperature controlled device, DVC1310 digital video camera, andQED Camera with Standalone 145 software. SDF-1α (100 ng/ml) was addedinto the culture after 6 hours and images were recorded for another 10hours. Digital video images were saved every 90 seconds, and cellmovement or morphological changes were analyzed with the NIH ImageAnalysis program. For movement analysis, the position of cell centroidwas measured every 15 minutes and plotted to show the trace of centroidmovement. The distance that the cell centroid transversed for each 90seconds was calculated to determine the speed of the movement. Formorphology analysis, the cell surface area and perimeter were measuredto represent the degree of rugged shape. The frequency and period offormation and retraction of filopodia and uropods were also analyzed.

Immunoblotting

Cells were lysed in lysis buffer (20 mM Tris pH 8.0, 150 mM NaCl, 10%glycerol, 1% NP-40, 0.42% NaF) containing protease inhibitors (1 mMPMSF, 1 mM Na₃VO₄, 5 μg/ml aprotinin, 5 μg/ml leupeptin). Cell lysateswere separated by 7.5% SDS-PAGE under reducing conditions andtransferred to nitrocellulose membranes (Schleicher & Schuell, Dassel,Germany). Proteins were detected by immunoblotting using an enhancedchemiluminescence technique (NEN Life Science Products, Mass.). Rabbitpolyclonal antibodies against c-Kit (C-19; Santa Cruz Biotechnology,Inc., Santa Cruz, Calif.), Akt, Akt (pSer 473), Akt (pThr 308), p70 S6kinase, and p70 S6 kinase (pThr 389) (Cell Signaling Technology, Inc.,Beverly, Mass.) and monoclonal antibodies against □-actin (AC-15; Sigma,St. Louis, Mo.) and phosphotyrosine (4G10, UBI, Lake Placid, N.Y.) wereused.

Example 1 CXCR4 is Ubiquitously Expressed and c-kit is VariablyExpressed in SCLC Cell Lines

The expression of chemokine receptor mRNAs in SCLC cells was determinedby RPA. Yeast tRNA and human control RNA-2 were used as the negative andpositive controls for CXCR4, respectively. All of the SCLC cell lines(10 total) tested expressed CXCR4 mRNA at various levels, with nodetectable mRNA for other chemokine receptors. The humanmegakaryoblastic cell line MO7e, as a control, expressed CXCR3 as wellas CXCR4. Expression of CXCR4 protein was confirmed by flow cytometricanalysis in these SCLC cell lines and MO7e. The expression of CXCR4 washighest in NCI-H209 and NCI-H446 cells. The expression of c-Kit in thesecell lines was evaluated by immunoblotting. 6 out of 10 SCLC cell linestested expressed variable levels of c-Kit (lines H69, H128, H209, H345,H510, and H526), with high expression in the MO7e control cells. Theblots were stripped and reprobed with an antibody against β-actin.

Example 2 SCF and SDF-1α Induce Proliferation of NCI-H69 Cells

The effect of SCF and SDF-1α on viability in NCI-H69 cells was analyzed.As described above in Example 1, NCI-H69 cells express both c-Kit andCXCR4, and thus these cells were used for many of the experiments.Without serum, neither SCF nor SDF-1α showed any effect on cellsurvival. On the other hand, in media containing 10% FCS, cellproliferation was significantly induced by SCF (21.5%, p=0.0373) andSDF-1α (26.6%, p=0.0133) separately or in combination (26.6%, p=0.0133)at 48 hours, as compared with untreated control. SCF and SDF-1α alsoconferred an increase in viable cell number at 72 hours (16.5%, p=0.0164and 15.5%, p=0.0184, respectively, and 20.0%, p=0.0322, combined). Eventhough SCF and SDF-1α individually induced proliferation of NCI-H69cells, there was no additive or synergistic effect seen with both thecytokine and chemokine combined. Similar results were observed fordifferent concentrations of FCS tested (0.5, 1, and 5% FCS), implicatingthe importance of SCF and SDF-1α in proliferation of NCI-H69 SCLC cells.

Example 3 SDF-1α Regulates Adhesion, Motility, and Cell Shape inNCI-H446 SCLC Cells

Cytoskeletal functions such as increased cell motility, adhesion toextra cellular matrix (ECM) proteins, morphological change, andmovement, are crucial for cancer cells to metastasize. To determine theeffect of SDF-1α on cell motility and adhesion, NCI-H446 cells thatexpress high amounts of CXCR4 and grow in an anchorage dependent fashionwere used. In an adhesion assay, FN (3.84-fold, p=0.0002) and col. IV(2.98-fold, p=0.0124) were found to increase the adhesion of NCI-H446cells as compared to the uncoated surface. In conjunction, SDF-1αstimulation further increased the attachment 3.14-fold on the uncoatedsurface (p<0.0001), but did not significantly enhance FN- and col.IV-mediated adhesion. In addition to cell adhesion, SDF-1α also markedlyincreased the motility and speed of NCI-H446 cells. The speed of cellmovement was calculated using the distance that each cell centroidtraversed per each 90 seconds. Morphological changes from round topolygonal shape including formation of neurite like projections,increased membrane ruffling, and more frequent filopodia and uropodsformations were observed in response to SDF-1α. Filopodia formation inthe presence of SDF-1α occurred much more frequently (13.14 vs. 2.86times/hour/cell) and for longer periods of time (6.09 vs. 3.75minutes/filopodium). Uropod formation was observed in 4 out of 7 (57.1%)SDF-1α treated cells. However, only 1 out of 7 (14.3%) untreated cellsshowed uropods. The period of time each uropod lasted also became muchlonger when induced by SDF-1α stimulation (12.2 vs. 5.0 minutes/uropod).

Example 4 PI3-K Regulates SDF-1α Induced Cell Motility of NCI-H446 SCLCCells

This example describes experiments conducted to determine whether thePI3-K inhibitor LY294002 can inhibit SDF-1α induced cell motility inNCI-H446 SCLC cells. NCI-H446 cells were either left untreated ortreated with SDF-1α (100 ng/ml) in the absence or presence of LY294002(25 μM). Phase-contrast microscopy pictures were taken at the 24 hourtime point. Most of the untreated cells kept their rounded shape andformed clusters, and nearly half of them attached weakly to the bottomof the dish. With SDF-1α, almost all cells tightly adhered to the bottomof the dish and neurite like projections were induced in many cells. Inthe presence of LY294002, more than 90% of the cells were floating andhad rounded shapes in spite of SDF-1α treatment, although they couldform clumps.

Example 5 CXCR4 and c-KIT Cooperatively Induce Morphological Changes inNCI-H69 SCLC Cells

It has not been shown previously that there is a functional interactionbetween CXCR4 and c-Kit in SCLC. NCI-H69 cells positive for both CXCR4and c-Kit expression were either untreated or treated with SDF-1α (100ng/ml) and/or SCF (100 ng/ml), in the absence or presence of STI571 (5μM). Phase-contrast microscopy pictures were then taken after 8 hours.Morphological changes began to be apparent at 4 hours, and the changesplateaued between 8 and 24 hours. Neurite like actin formations wereobserved in response to SCF, and this morphological change was moreapparent as projections when the cells were treated with SDF-1α. Neuritelike projections induced by SDF-1α alone still formed even in thepresence of STI571. STI571 treated NCI-H69 cells stimulated with bothSDF-1α and SCF could not form any neurite like structures. STI571inhibited the morphological changes only in the presence of SCF. Theseresults suggest that there are important interactions between CXCR4 andc-Kit in SCLC that influence cell motility. Inhibition of the activec-Kit receptor by STI571 may lead to the inhibition of other activereceptors such as CXCR4.

Example 6 SDF-1α and SCF Independently Regulate Phosphorylation of AKTand P70 S6 Kinase

PI3-K is important in the regulation of cytoskeletal functions in SDF-1αand SCF signaling (Ganju, R. K. et al. (1998) J. Biol. Chem.273:23169-75; Linnekin, D. et al. (1999) Int. J. Biochem. Cell Biol.31:1053-74; Vicente-Manzanares, M. et al. (1999) J. Immunol.163:4001-12; Wang, J. F. et al. (2000) Blood 95:2505-13; Zhang, X. F. etal. (2001) Blood 97:3342-8). The activity of the PI3-K downstreamtargets Akt and p70 S6 kinase is regulated through criticalserine/threonine (Ser/The) residues (Franke, T. F. et al. (1997) Cell88:435-7; Pullen, N. and Thomas, G. (1997) FEBS Lett 410:78-82). Serumstarved NCI-H69 cells were stimulated with SDF-1α (50 ng/ml) or SCF (50ng/ml) for 0-60 minutes before lysis. Cell lysates were applied to a7.5% SDS-PAGE gel and transferred to nitrocellulose membrane. Themembrane was probed with monoclonal antibodies against phosphotyrosine(4G10) and β-actin and polyclonal antibodies against Akt (pSer 473) andp70 S6 kinase (S6K) (pThr 389). Both SDF-1α and SCF induced timedependent tyrosine phosphorylation of cellular proteins and Ser/Thrphosphorylation of Akt and p70 S6 kinase in NCI-H69 cells. Severaltyrosine phosphorylated bands were identified between 70-120 kDa within15 minutes of SDF-1α stimulation. On the other hand, maximal tyrosinephosphorylation of proteins at 60-90 kDa and 110-145 kDa occurred within2.5-7.5 minutes in response to SCF. Dose-response studies indicated thatoptimal phosphorylation of cellular proteins was obtained with at least25 ng/ml SDF-1α and 10 ng/ml SCF. Phosphorylation of Akt (Ser 473) andp70 S6 kinase (Thr 389) occurred in response to SDF-1α within 5 minutesand in response to SCF within 2.5 minutes.

Example 7 STI571 and LY294002 Inhibit Signal Transduction of CXCR4 andc-Kit Pathways

The small molecular inhibitors STI571 (which targets c-Kit) and LY294002(which targets PI3-K) were utilized to determine the effects ondownstream signaling by SDF-1α and SCF in NCI-H69 cells. Cells were leftuntreated or pre-treated with STI571 (5 μM) or LY294002 (25 μM)overnight in serum starved media and subsequently stimulated with 50ng/ml SCF and/or 50 ng/ml SDF-1α for 15 minutes before lysis. Lysateswere processed as described above in Example 6, and the membrane wasprobed with the phospho-specific or regular antibodies against Akt andp70 S6 kinase, as well as with an anti-c-Kit antibody. Cooperativephosphorylation of Akt at Ser 473/Thr 308 and p70 S6 kinase at Thr 389was induced by SCF and SDF-1α. STI571 pre-treatment inhibited SCF butnot SDF-1α induced phosphorylation. The expression levels of Akt, p70 S6kinase, and c-Kit were not affected by any of these treatments. Incontrast, LY294002 pre-treatment blocked SDF-1α as well as SCF inducedphosphorylation of Akt and p70 S6 kinase.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method of inhibiting cellular proliferation in a small cell lungcancer cell population comprising contacting the population with a CXCR4inhibitor.
 2. A method of inhibiting cellular movement or motility in asmall cell lung cancer cell population comprising contacting thepopulation with a CXCR4 inhibitor.
 3. A method of modulating cellularadhesion in a small cell lung cancer cell population comprisingcontacting the population with a CXCR4 inhibitor.
 4. A method ofinhibiting morphological change in a small cell lung cancer cellpopulation comprising contacting the population with a CXCR4 inhibitor.5. The method of claim 1, further comprising contacting the small celllung cancer cell population with a receptor tyrosine kinase inhibitor.6. The method of claim 5, wherein the receptor tyrosine kinase inhibitoris a c-Kit inhibitor.
 7. A method of treating a subject with small celllung cancer comprising administering a CXCR4 inhibitor to the subject.8. The method of claim 7, further comprising administering a receptortyrosine kinase inhibitor to the subject.
 9. The method of claim 8,wherein the receptor tyrosine kinase inhibitor is a c-Kit inhibitor. 10.A method of inhibiting metastasis of small cell lung cancer in a subjectcomprising administering a CXCR4 inhibitor to the subject.
 11. Themethod of claim 10, further comprising administering a receptor tyrosinekinase inhibitor to the subject.
 12. The method of claim 1 1, whereinthe receptor tyrosine kinase inhibitor is a c-Kit inhibitor.
 13. Amethod for identifying an agent which can be used to treat small celllung cancer comprising determining whether the agent inhibits CXCR4. 14.A method for determining whether a CXCR4 inhibitor can or cannot be usedto treat small cell lung cancer comprising: a) obtaining a sample oflung cancer cells; and b) determining whether the cells express CXCR4;thereby determining that the CXCR4 inhibitor can be used to treat smallcell lung cancer when CXCR4 is expressed.
 15. A method for determiningwhether a combination of a CXCR4 inhibitor and a receptor tyrosinekinase inhibitor can or cannot be used to treat small cell lung cancercomprising: a) obtaining a sample of lung cancer cells; and b)determining whether the cells express CXCR4 and the receptor tyrosinekinase that is inhibited by said receptor tyrosine kinase inhibitor;thereby determining that the combination of a CXCR4 inhibitor and areceptor tyrosine kinase inhibitor can be used to treat small cell lungcancer when CXCR4 and the receptor tyrosine kinase are expressed. 16.The method of claim 15, wherein the receptor tyrosine kinase inhibitoris a c-Kit inhibitor.
 17. A method for determining whether a CXCR4inhibitor can or cannot be used to inhibit proliferation or metastasisof small cell lung cancer comprising: a) obtaining a sample of lungcells; and b) determining whether the cells express CXCR4; therebydetermining that the CXCR4 inhibitor can be used to inhibitproliferation or metastasis of small cell lung cancer when CXCR4 isexpressed.
 18. A method for determining whether a combination of a CXCR4inhibitor and a receptor tyrosine kinase inhibitor can or cannot be usedto inhibit proliferation or metastasis of small cell lung cancercomprising: a) obtaining a sample of lung cells; and b) determiningwhether the cells express CXCR4 and the receptor tyrosine kinase that isinhibited by said receptor tyrosine kinase inhibitor; therebydetermining that the combination of a CXCR4 inhibitor and a receptortyrosine kinase inhibitor can be used to inhibit proliferation ormetastasis of small cell lung cancer when CXCR4 and the receptortyrosine kinase are expressed.
 19. The method of claim 18, wherein thereceptor tyrosine kinase inhibitor is c-Kit inhibitor.
 20. A method fordetermining whether a patient would benefit from treatment with an agentthat inhibits CXCR4 comprising: a) obtaining a lung sample from thepatient; and b) determining whether CXCR4 is expressed in the sample.21. A method for determining whether a patient would benefit fromtreatment with an agent that inhibits CXCR4 and an agent that inhibitsc-Kit comprising: a) obtaining a lung sample from the patient; b)determining whether CXCR4 is expressed in the sample; and c) determiningwhether c-Kit is expressed in the sample.
 22. A method for determiningwhether treatment with a CXCR4 inhibitor should be continued ordiscontinued in a small cell lung cancer patient, comprising: a)obtaining two or more samples comprising tumor cells from a patientduring the course of treatment; b) determining the level of activity inthe tumor cells of CXCR4; and c) continuing treatment when the activityof CXCR4 does not increase during treatment.
 23. A method fordetermining whether treatment with a combination of a CXCR4 inhibitorand a receptor tyrosine kinase inhibitor should be continued ordiscontinued in a small cell lung cancer patient, comprising: a)obtaining two or more samples comprising tumor cells from a patientduring the course of treatment; b) determining the level of activity inthe tumor cells of CXCR4; c) determining the level of activity in thetumor cells of c-Kit; and d) continuing treatment when the activitylevels of CXCR4 and/or c-Kit do not increase during treatment.
 24. ACXCR4 inhibitor for use in a method for the treatment of a subject. 25.Use of a CXCR4 inhibitor for the preparation of a pharmaceuticalcomposition for use in a method such as inhibiting cellularproliferation in a small cell lung cancer cell population, inhibitingcellular movement or motility in a small cell lung cancer cellpopulation, modulating cellular adhesion in a small cell lung cancercell population, inhibiting morphological change in a small cell lungcancer cell population, treating a subject having small cell lungcancer, or inhibiting metastasis of small cell lung cancer in a subject.26. A combination which comprises (a) a CXCR4 inhibitor and (b) areceptor tyrosine kinase inhibitor, such as especially a c-Kitinhibitor, wherein the active ingredients (a) and (b) are present ineach case in free form or in the form of a pharmaceutically acceptablesalt, for simultaneous, concurrent, separate or sequential use,especially in a method for the treatment of a subject, preferably in amethod such as inhibiting cellular proliferation in a small cell lungcancer cell population, inhibiting cellular movement or motility in asmall cell lung cancer cell population, modulating cellular adhesion ina small cell lung cancer cell population, inhibiting morphologicalchange in a small cell lung cancer cell population, treating small celllung cancer, or inhibiting metastasis of small cell lung cancer.